Mycobacterial Gene cuvA Is Required for Optimal Nutrient Utilization and Virulence

1

The mycobacterial gene cuvA/Rv1422 is required for optimal nutrient utilization and 1

virulence 2

Mushtaq Mir1*+, Sladjana Prisic1+, Choong-Min Kang1**, Shichun Lun2, Haidan Guo2, Jeffrey P. 3

Murry3***, Eric J. Rubin3, Robert N. Husson1# 4

5

1Division of Infectious Diseases, Children’s Hospital, Harvard Medical School, Boston, MA, 6

02115 7

2Center for Tuberculosis Research, Johns Hopkins University School of Medicine, Baltimore, 8

MD, 21231 9

3Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, 10

MA 02115 11

*Current address: Division of Genetics, Wadsworth Center, Center for Medical Science, New 12

York State Department of Health, Albany, NY 12201 13

**Current address: Department of Biological Sciences, California State University Stanislaus, 14

Turlock, CA 95382 15

***Current address: Gilead Sciences, 333 Lakeside Drive, Foster City, CA 94404 16

+These authors contributed equally to this work. 17

Running title: Mycobacterial cuvA: nutrient utilization and virulence 18

Key words: Mycobacterium, cholesterol, bacterial cell pole, peptidoglycan synthesis, virulence, 19

cell wall, Rv1422 20

#Corresponding author information: 21 Robert N. Husson 22 Division of Infectious Diseases 23 Children’s Hospital Boston 24 300 Longwood Ave. 25 Boston, MA 02115 26 Tel 617-919-2900/Fax 617-730-0254 27 [email protected] 28

IAI Accepts, published online ahead of print on 21 July 2014Infect. Immun. doi:10.1128/IAI.02207-14Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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Abstract 29

To persist and cause disease in the host, Mycobacterium tuberculosis must adapt to its 30

environment during infection. These adaptations include changes in nutrient utilization and 31

alterations in growth rate. M tuberculosis Rv1422 is a conserved gene of unknown function that 32

was found in a genetic screen to interact with the mce4 cholesterol uptake locus. Rv1422 33

protein is phosphorylated by the M. tuberculosis Ser/Thr kinases PknA and PknB, which 34

regulate cell growth and cell wall synthesis. Bacillus subtilis strains lacking the Rv1422 35

homologue yvcK grow poorly on several carbon sources, and yvcK is required for proper 36

localization of peptidoglycan synthesis. Here, we show that M. smegmatis and M. tuberculosis 37

strains lacking Rv1422 have growth defects in minimal medium containing limiting amounts of 38

several different carbon sources. These strains also have morphologic abnormalities, including 39

shortened and bulging cells, findings that suggest a cell wall defect. In both mycobacterial 40

species, Rv1422 localizes uniquely to the growing cell pole, the site of peptidoglycan synthesis 41

in mycobacteria. M. tuberculosis ΔRv1422 is markedly attenuated for virulence in a mouse 42

infection model, where it elicits decreased inflammation in the lungs and shows impaired 43

bacterial persistence. These findings lead us to name this gene cuvA (carbon utilization and 44

virulence protein A) and suggest a model in which deletion of cuvA leads to changes in nutrient 45

uptake and/or metabolism that affect cell wall structure, morphology and virulence. Its role in 46

virulence suggests that CuvA may be a useful target for novel inhibitors of M. tuberculosis 47

during infection. 48

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Introduction 50

Regulated pathways that allow physiologic adaptations in response to changes in the 51

environment are critical for the growth and survival of all bacteria. In the case of the human 52

pathogen Mycobacterium tuberculosis, where infection can persist for decades and cause 53

several different disease manifestations, the bacterium must adapt to multiple environments 54

during the prolonged course of infection. These environments include intracellular milieus within 55

host phagocytic cells such as neutrophils, macrophages and dendritic cells that may be part of 56

organized granulomas, as well as in extracellular environments such as occur in caseating 57

granulomas. In addition to mechanisms to defend against and modulate host antibacterial 58

activities, a critical adaptation of M. tuberculosis during infection is utilization of host lipids as a 59

primary carbon source (1-3). Another key bacterial adaptation during M. tuberculosis infection is 60

regulation of replication, which changes from rapid growth during the first few weeks of infection, 61

to markedly reduced bacterial replication following the onset of adaptive immunity (3, 4). 62

Gaining insight into bacterial adaptive mechanisms is critical for understanding tuberculosis 63

pathogenesis and may lead to novel therapeutic approaches that target these bacterial adaptive 64

processes. 65

M. tuberculosis Rv1422 encodes a protein of unknown function that has homologues in 66

other mycobacteria and across a broad range of bacterial phyla. Our interest in this protein as 67

potentially having an important role in bacterial adaptation during infection was motivated by 68

three observations. One is our previous finding that it is phosphorylated in M. tuberculosis cells 69

by the Ser/Thr protein kinases PknA and PknB. These kinases are essential for growth and 70

regulate cell division, peptidoglycan (PGN) synthesis and cell morphology (5-9), suggesting that 71

Rv1422 may play a role in one or more of these processes. Second, Rv1422 was identified in a 72

genome-wide transposon screen for genetic interactions with the mce1 and mce4 loci. The 73

mce4 locus encodes a cholesterol uptake system (2, 10) and the mce1 locus has been 74

implicated in the transport of other lipids (11), suggesting that the Rv1422 protein may function 75


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in utilization of cholesterol or other lipids. In addition, an M. tuberculosis strain with transposon 76

insertion in Rv1422 was defective for replication in mouse spleens in an 8-week infection model, 77

suggesting that Rv1422 might also be important in M. tuberculosis virulence (12). 78

The orthologue of Rv1422 has been investigated in the model organism Bacillus subtilis, 79

where a null mutant of this gene, yvcK, was found to have partial growth defects on media 80

containing several different carbon sources, including tricarboxylic acid cycle intermediates (13). 81

On transfer to medium containing carbon sources in which this organism grew poorly, bulging 82

and lysis of cells were seen, indicating perturbation of PGN synthesis. Subsequently, YvcK was 83

found to be distributed in a circumferential pattern around the long axis of the cell in B. subtilis, 84

paralleling the distribution of PGN synthesis (14). Overexpression of MreB, a filament-forming 85

protein that is similarly distributed around the circumference of the cell (15), was shown to 86

suppress the carbon-source specific growth and morphology defects of the yvcK null mutant. In 87

addition, the PGN synthesis protein PBP1 was found to be mis-localized in the ΔyvcK strain in a 88

carbon-source-dependent manner (14). Thus, though its mechanism of action is not known, 89

YvcK appears to be required for proper localization of PGN synthesis and production of a 90

normal cell wall in B. subtilis. 91

These data from M. tuberculosis and B. subtilis led us to ask whether Rv1422 plays a 92

role in carbon source utilization, cell growth, and virulence in mycobacteria. Here we identify 93

growth defects on multiple carbon sources of deletion mutants in M. tuberculosis Rv1422 and 94

the M. smegmatis homologue MSMEG_3080 compared to wild type. Because of the importance 95

of cholesterol as a carbon source for M. tuberculosis during infection (2, 16, 17), we used 96

cholesterol as a representative carbon source for additional investigation of the function of 97

Rv1422. When grown on cholesterol, both the M. smegmatis and M. tuberculosis mutant strains 98

have morphologic abnormalities including bulging and shortened cell length, indicating a role for 99

Rv1422 in cell wall synthesis. We also show striking localization of Rv1422-gfp fusions 100

exclusively to the growing cell pole, the site of PGN synthesis in mycobacteria (8) and 101


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demonstrate that the M.smegmatis deletion strain are hypersusceptbible to several β-lactam 102

antibiotics when grown on cholesterol, findings that link Rv1422 to cell growth and cell wall 103

synthesis. Consistent with these in vitro phenotypes, the virulence of the M. tuberculosis mutant 104

is markedly attenuated in a mouse infection model. Based on these results, we have named 105

this gene cuvA (carbon-source utilization and virulence protein A). 106

Materials and Methods 107

Strains, media, plasmids and primers 108

M. tuberculosis H37Rv and M. smegmatis mc2-155 (18) were used as wild type strains 109

and are the parental strains in which mutants were made. E. coli TOP10 (Invitrogen) was used 110

for cloning and was grown in LB broth. For routine growth, M. tuberculosis or M. smegmatis 111

were grown at 37o C in Middlebrook 7H9 liquid medium (Difco) supplemented with 0.5% 112

albumin, 0.2% glucose, 0.085% NaCl, 0.2% Glycerol and 0.05% Tween 80 (7H9-ADC-Tw). 113

Kanamycin (20 μg ml-1) or hygromycin (50 μg ml-1) was added to liquid or agar medium when 114

appropriate. Details of primers, plasmids and strains are shown in Table S1. 115

For growth of M. smegmatis on different carbon sources in liquid medium, cells were 116

grown in minimal medium (MM) (1 g/L KH2PO4, 2.5 g/L Na2HPO4, 0.5 g/liter asparagine, 0.5 g/L 117

MgSO4x7H2O, 0.5 mg/L CaCl2, 0.1 mg/L ZnSO4, 50 mg/L ferric ammonium citrate and 0.05% 118

Tyloxapol) (2), containing 0.01% (wt/vol) of glucose, glycerol, cholesterol, propionate, gluconate 119

or citrate. MM plus 0.2% glucose was used as a positive control for maximal growth. For 120

measuring growth on solid medium, cells grown in Middlebrook 7H9 liquid medium to an OD600 121

of 0.5 were washed in PBS containing 0.05% Tyloxapol and 10-fold serial dilutions in PBS were 122

spotted on agar plates containing MM as described above (without tyloxapol) plus 0.01% of the 123

carbon sources. MM plates containing 0.2% glucose were included as a control. Plates were 124

incubated at 37o C and photographed after one month at 37oC for M. tuberculosis and after 2 125

days for M. smegmatis. 126


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For subcellular localization, CuvA was expressed as a carboxy-terminal GFP fusion. 127

cuvA was PCR-amplified from genomic DNA of M. tuberculosis H37RV using primers 128

Rv1422AE and Rv1422rPac1. gfp was PCR-amplified from pTracerCMV (Invitrogen) using 129

primers GFP3PacI and GFP4XK. Overlap PCR was then carried out using primers Rv1422AE 130

and GFP4XK giving rise to the fusion PCR product Rv1422-gfp. The Rv1422-gfp PCR product 131

was digested with AseI and XbaI (the AseI site in the Rv1422 gene was eliminated by overlap 132

PCR mutagenesis to create a silent mutation) and cloned into the integrating vector pMV306AC 133

3’ of the acetamidase promoter to obtain pMV306Ac-Rv1422-gfp (pMV306Ac-cuvA-gfp). 134

For localization of M. smegmatis PBP1 (encoded by MSMEG_6900), a C-terminal 135

MSMEG_6900-RFP fusion protein was expressed. The MSMEG6900 gene was amplified by 136

PCR from M. smegmatis genomic DNA using primers MSMEG6900-F and MSMEG6900-R. 137

Similarly rfp was PCR-amplified using primers Rfp_F and Rfp_R. Overlap PCR was then carried 138

out using the primers MSMEG6900-F and Rfp_R. The resulting fusion PCR product was cloned 139

into the NdeI and XbaI sites in pMV261Ac 3’ of the acetamidase promoter to obtain pMV261Ac-140

MSMEG6900-rfp. All recombinant clones used in this study were sequenced to rule out any 141

mutations. 142

Generation of M. smegmatis and M. tuberculosis cuvA deletion strains 143

To delete cuvA (MSMEG_3080) in M. smegmatis, a 1008 bp region (L-arm) 5’ to 144

MSMEG_3080 including 6 codons of MSMEG_3080 was PCR-amplified using primers 145

MSMEG3080-cond-1 and MSMEG3080-cond-2. Similarly a 1040 bp region (R-arm) 3’ to 146

MSMEG_3080 including 100 bp of the MSMEG_3080 gene was PCR-amplified using primers 147

MSMEG3080-cond-3 and MSMEG3080-cond-4. Overlap PCR was then carried out with primers 148

MSMEG3080-cond-1 and MSMEG3080-cond-4. The PCR product contained a PacI site 149

between the arms and was cloned into pRH1351 (19). A hygromycin resistance cassette was 150

introduced at the PacI site and the targeting construct thus obtained was electroporated into M. 151

smegmatis mc2155. Candidate deletion strains were obtained using a two-step double 152


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counterselection method, as previously described (19, 20). PCR of genomic DNA from 153

candidate mutant strains, using primers annealing to flanking regions of MSMEG_3080, yielded 154

the expected 1.5 kb PCR product, confirming the replacement of MSMEG_3080 (cuvA) in the 155

genome by the hygromycin cassette (Fig. S1). For complementation of the M. smegmatis cuvA 156

deletion strain, cuvA was PCR-amplified using primers MSMEG3080-int-1 and MSMEG3080-157

int-2 and cloned under control of the tet promoter in pMind (21). This complemented strain was 158

grown in the presence of 5 ng/ml tetracycline to induce expression of cuvA. 159

The cuvA (Rv1422) gene was deleted in M. tuberculosis H37Rv genome using similar 160

methods. One kb regions 5’ and 3’ to Rv1422 were PCR-amplified and ligated to the 5’ and 3’ 161

ends respectively of a hygromycin-chloramphenicol cassette. The ligated DNA fragment was 162

introduced into a temperature-sensitive mycobacterial suicide vector harboring sacB and xylE 163

genes. The targeting vector was transformed into wild type M. tuberculosis H37RV and 164

candidate deletion strains were obtained by counterselection on sucrose-containing plates at 165

38oC. Deletion of cuvA was confirmed by PCR of genomic DNA using primers annealing to 166

flanking regions of the gene. The expected size of 2.1 kb resulting from the replacement of cuvA 167

by the hygromycin-chloramphenicol cassette confirmed the deletion of the gene (Fig. S1). The 168

complementing construct was obtained by cloning cuvA downstream of the hsp70 promoter in 169

the integrating vector pJEB402 (22). 170

Microplate Alamar Blue assay (MABA) for growth 171

M. smegmatis strains were shaken at 37oC in 7H9-ADC-Tw (without glycerol) until they reached 172

OD600 ~0.5. The cells were spun down, washed twice in PBS-Tx (phosphate buffered saline, 173

0.05% Tyloxapol) and diluted to calculated OD600 = 0.001 in MM alone or with added 0.2% 174

glucose, or each of the following added to 0.01%: glucose, cholesterol, glycerol, gluconate, 175

citrate or propionate. Tetracycline (5 ng/mL) was present in all cultures. 200 µl of each culture 176

was placed in a 96-well clear bottom plate and 20 µl filtered Alamar Blue (Life Technologies) 177

was added. Fluorescence (Excitation/Emission = 550nm/590nm) was measured every hour 178


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from the bottom of the well in a plate reader (Tecan) with incubation at 37oC. The exponential 179

portion of the growth curves were analyzed by non-linear curve fitting to an exponential growth 180

model and the rate constants were compared by an extra sum of squares F test, using 181

Graphpad Prism 5.0 software. 182

MABA susceptibility determination 183

M. smegmatis cultures were prepared as for the MABA growth curves described above, 184

except that they were diluted to a calculated of OD600 of 0.005 in MM containing 0.01% 185

cholesterol. Glucose was added to half of the cultures, to obtain cultures with 0.01% cholesterol 186

plus 0.2% glucose; the other cultures contained 0.01% cholesterol only. To half of the 187

cholesterol alone cultures and half of the cholesterol plus glucose cultures, clavulanate was 188

added to a final concentration of 2.5 µg/ml. For each antibiotic tested, two-fold dilutions were 189

made and M. smegmatis cultures (190 µl) and antibiotics (10 µl) were mixed in clear 96-well 190

plates. After overnight (for glucose + cholesterol) or 2 days (for cholesterol only) incubation, 50 191

µl of 0.5X Alamar Blue reagent diluted in 10% Tween 80 was added. After overnight incubation 192

fluorescence (Ex/Em = 550nm/590nm) was measured. Growth inhibition was calculated using 193

the following formula: 1 100, where Fmin is the 194

fluorescence from wells without growth and Fmax is the fluorescence from control wells that did 195

not contain any antibiotic. The MIC90 was defined as the concentration of antibiotic that caused 196

90% growth inhibition (23). 197

M. tuberculosis H37Rv was tested for antibiotic susceptibility in a similar way, except 198

that cultures were pre-incubated in PBS-Tx for 2 days and were diluted to OD600 0.05, and the 199

clavulanate concentration was 7.5µg/ml. Plates were incubated for one week before Alamar 200

blue was added and analyzed as described above. The final concentration ranges of the 201

antibiotics tested were: rifampin (M. tuberculosis), 0.003-0.64 μg/ml; rifampin (M. smegmatis), 202

0.3-80 μg/ml; levofloxacin, 0.01-2.56 μg/ml; ampicillin, 4-1000 μg/ml; cefotaxime, 4-1000 μg/ml; 203


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cephalothin, 2-500 μg/ml; meropenem, 0.2-50 μg/ml. For both M. smegmatis and M. 204

tuberculosis, each antibiotic was tested in at least three biological replicates with technical 205

duplicates for every experiment. MICs between strains were compared using the t-test in 206

Microsoft Excel. 207

Microscopy and fluorescent vancomycin staining 208

For cellular localization of CuvA the recombinant clone pMV306-pacet-Rv1422-gfp was 209

transformed into M. smegmatis strain mc2155 and in to an M. tuberculosis leucine/pantothenate 210

double auxotrophic strain (18, 24). The pMV306-pacet-gfp expressing GFP alone was 211

transformed into M. smegmatis as a control. The M. smegmatis strains were grown to an OD600 212

of 0.1 - 0.2 in 7H9 broth, induced with 0.2% acetamide for 6-8 hrs and visualized by microscopy. 213

The M. tuberculosis strains were grown to an OD600 of 0.5 and inoculated onto a layer of nutrient 214

agarose medium on microscope slides (agarose pads) made with Middlebrook 7H9 medium 215

containing glucose and 0.2% acetamide. Cells on these agarose pads were observed at 24 216

hours after growth. 217

For staining sites of peptidoglycan synthesis, Vancomycin-Alexa 568 (Molecular Probes) 218

was prepared and used as previously described (8). Vancomycin-Alexa568 was added to 219

cultures at OD600 of 0.1 - 0.2 to achieve a final concentration of 5 μg/ml and growth was 220

continued further for 2.5 h in the dark. The cells were washed, transferred onto a glass slide, air-221

dried, and a cover slip was mounted using Prolong Gold anti-fade reagent (Invitrogen). Cells 222

were observed using Zeiss AxioImager.Z2 microscope with 63x differential interference contrast 223

oil immersion objective. For fluorescence, green and red fluorescence filters were used. Images 224

were captured by a CoolSNAP HQ2 camera (Photometrics), acquired with AxiVision 4.8 225

software and processed by Adobe Photoshop CS5. 226

For examination of cell morphology, cells were grown in the indicated medium, pelleted 227

and fixed in 4% paraformaldehyde prepared in PBS for 2 hours. Ammonium chloride was then 228


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added to a final concentration of 50 mM, cells were pelleted and then resuspended in PBS. 5 �l 229

of the cell suspension was spotted on a glass slide, which was air-dried, mounted with a 230

coverslip and observed using either a 63x or 100x differential interference contrast oil immersion 231

objective. Images were processed using Adobe Photoshop CS5. Measurement of cell length 232

was performed using ImageJ software and statistical analysis of cell length was performed with 233

one-way ANOVA and the nonparametric Kruskal-Wallis test using Graphpad Prism software. 234

For examination of live M. smegmatis, cells were grown overnight on agarose pads on glass 235

microscope slides. The cells were then directly observed on the agarose pads. 236

Transposon Mutagenesis 237

To identify mutations that suppress the cholesterol growth defect of the ΔcuvA strain of 238

M. tuberculosis, we performed transposon mutagenesis using the φMycoMarT7 phagemid (25, 239

26). Preparation of the phage stock, its titration and subsequent transduction of cuvA deletion 240

strain was carried out as described (27). Approximately 120,000 transductants were plated on 241

cholesterol-agar plates containing 20 μg/ml kanamycin and incubated at 37oC. After 5 weeks, 242

60 colonies were streaked on cholesterol-agar plates, of which 20 showed substantial growth. 243

Genomic DNA was isolated and three restriction enzymes: BamHI, SphI and SacII were used 244

individually to completely digest the genomic DNA. The recognition sites of these enzymes are 245

absent in the transposon. The fragmented DNA was purified and treated with T4 DNA ligase to 246

circularize the fragments. PCR amplification was then carried out using outward primers 247

annealing to the ends of transposon. The PCR products were analyzed by agarose gel 248

electrophoresis and strong intensity bands were extracted and sequenced to identify the site of 249

transposon insertion. 250

Mouse Infection experiments 251

Mouse infection experiments were performed at the NIAID-sponsored Tuberculosis 252

Animal Research and Gene Evaluation Task Force (TARGET). Mouse infection protocols were 253


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approved by the Animal Care and Use Committee at Johns Hopkins School of Medicine, 254

Baltimore, MD and all experiments involving mice were carried out according to these protocols. 255

To determine the effect of cuvA deletion on growth of M. tuberculosis in vivo, wild type 256

H37Rv, the cuvA deletion strain (RH 478) and the ΔcuvA complemented strain (RH480), were 257

used to perform aerosol infection of BALB/c mice. Prior to infection, all M. tuberculosis stains 258

were confirmed to produce phthiocerol dimycolate and to have positive neutral red staining 259

(data not shown). For time to death experiments strains were grown to mid-log phase and 260

diluted to OD600 of 0.2 in 10 ml of 7H9 Middlebrook liquid medium supplemented with OADC. 16 261

BALB/c mice per strain were infected for 30 min in a Glas-col aerosol machine. At 1 day post 262

infection 3 mice per strain were sacrificed. Lungs from each mouse were homogenized and 263

dilutions of lung homogenate were plated on 7H11 antibiotic selective plates. CFU were 264

recorded after 3-4 weeks of incubation at 37oC. The remaining 12 mice infected with each strain 265

were monitored to record the days to death or severe illness for each mouse. Statistical analysis 266

of survival was performed with the Log Rank (Mantel-Cox) test using GraphPad Prism 5.0 267

software. 268

To determine the CFU of each strain in lung and spleen following infection, the three 269

strains were grown to an OD600 of 0.05 to 0.1 in Middlebrook 7H9 liquid medium supplemented 270

with OADC. 24 BALB/c mice per strain were infected as described above. Four mice per strain 271

were sacrificed at day 1, 14, 28, 56, 84 and 112 following infection. The lung and spleen of 272

each mouse was weighed and gross pathology pictures were taken. 3/4 of each lung and 273

spleen was homogenized and dilutions were plated on 7H11 selective plates. The CFU were 274

counted after 3-4 weeks at 37oC. The remaining 1/4 of each lung and spleen was stained with 275

hematoxylin and eosin for histopathology. CFU in lungs, lung weight and spleen weight for each 276

strain were compared at the indicated time points by one-way ANOVA with Tukey’s post-test 277

using GraphPad Prism 5.0 software 278

Results 279


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CuvA is required for optimal growth and normal cell morphology on several carbon 280

sources 281

Mutant strains containing cuvA deletions were constructed by allelic exchange in both 282

the pathogen M. tuberculosis and the rapid-growing non-pathogen M. smegmatis (Fig. S1). A 283

complemented M. tuberculosis ΔcuvA strain was constructed by expressing cuvA under control 284

of the hsp70 promoter in the vector pJEB402 (22), to allow stable expression during prolonged 285

incubation in vitro and during infection experiments. The same construct was used to 286

complement the M. smegmatis mutant with M. tuberculosis cuvA. To complement the M. 287

smegmatis mutant with the M. smegmatis cuvA homologue MSMEG_3080, this gene was 288

expressed under control of a Tet repressor-regulated promoter, to allow inducible expression of 289

this gene (21). 290

When grown in Middlebrook 7H9 liquid medium containing 0.2% glucose, M. 291

tuberculosis ΔcuvA (Fig. 1A) and M. smegmatis ΔcuvA (Fig. S4A) grew at a similar rate and to 292

the same final density as the wild type parental strains. Based on genetic data linking Rv1422 to 293

the Mce4 cholesterol transport system (28), we initially examined growth of the deletion strains 294

in minimal medium (MM) with cholesterol added as a carbon source. In contrast to the results 295

obtained with glucose-containing 7H9 medium, we observed altered growth phenotypes for both 296

the M. smegmatis and M. tuberculosis ΔcuvA strains when they were grown on MM plus 297

cholesterol, though these growth defects were not identical in the two species. 298

For M. tuberculosis, our attempts to compare growth by A600 measurements of wild type, 299

ΔcuvA and complemented strains in liquid MM containing 0.01% cholesterol were limited by 300

marked clumping of all strains and by the medium being cloudy and forming precipitates. We 301

therefore used an alternative measure of growth, the microplate Alamar Blue assay (MABA), 302

which measures dye reduction in metabolically active growing cells. Data from this assay did not 303

show a clear growth defect of M. tuberculosis ΔcuvA in liquid medium containing cholesterol, 304


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although all M. tuberculosis strains grew poorly and required a high inoculum to grow in this 305

medium, limiting interpretation of this result (Fig. S2). On solid medium, however, the M. 306

tuberculosis ΔcuvA strain had a clear growth defect compared to wild type (Fig. 1B). When 307

grown in Middlebrook 7H9 medium, washed and spotted onto agar plates, the wild type strain 308

grew and formed colonies on MM agar with either 0.01% cholesterol or 0.2% glucose, although 309

the growth on MM plus cholesterol was slower. The ΔcuvA strain grew more slowly than wild 310

type on MM plus 0.2% glucose and showed minimal growth on cholesterol, even after prolonged 311

incubation. These growth phenotypes were complemented by expression of cuvA in the M. 312

tuberculosis mutant. 313

The observation that the B. subtilis yvcK deletion had growth defects on multiple carbon 314

sources, led us to compare the growth of wild type M. tuberculosis to the cuvA deletion strain on 315

solid MM containing several other carbon sources. Each compound was added to a final 316

concentration of 0.01%, the concentration of cholesterol that has been used in mycobacterial 317

growth media because of its low aqueous solubility (2, 17). As shown in Figure 2, the M. 318

tuberculosis cuvA mutant strain grew much more slowly than wild type on each of these media, 319

as indicated by less dense growth and absence of visible growth at lower dilutions. In each case 320

the complemented strain showed growth similar to, though slightly less than that of the wild 321

type. These data indicate that the growth defect of the ΔcuvA strain is not specific, but rather is 322

a general defect in utilization of these several different carbon sources. 323

Because CuvA was shown to be phosphorylated in M. tuberculosis (5, 29), to determine 324

whether phosphorylation of CuvA is important for its function in nutrient utilization, 325

complementing constructs encoding CuvA in which the phosphoacceptor Thr residue was 326

replaced with either a non-phosphorylatable (Thr325Ala) or a phospho-mimetic (Thr325Glu) 327

amino acid were tested. Both of these mutant alleles also fully complemented the cholesterol 328


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growth defect (Fig. S3) suggesting that phosphorylation of Thr325 is not required for CuvA 329

function in cholesterol utilization under the conditions examined in these experiments. 330

We then examined growth of the M. smegmatis ΔcuvA strain. This strain had substantial, 331

statistically significant growth defects observed using the MABA assay when grown in liquid MM 332

alone or with several carbon sources present at 0.01%, though growth was similar to wild type 333

in MM plus glucose or gluconate (Fig. 3). The mutant growth defects were well-complemented 334

in each case by MSMEG_3080 expressed in trans. Addition of 0.2% glucose both increased the 335

growth rate and eliminated the difference between the wild type and mutant strains grown on 336

cholesterol or propionate (data not shown), indicating that the growth defect is not likely to be 337

due to toxic effect of defective cholesterol catabolism as has been observed in other mutants 338

that cannot utilize cholesterol (16). When grown on solid medium, the ΔcuvA grew less than wild 339

type on MM alone or with added cholesterol, but grew as well as wild type on 0.2 or 0.01% 340

glucose (Fig S4B). These data show that the growth defects of M. smegmatis ΔcuvA, like that of 341

M. tuberculosis, are not specific. 342

To further investigate the growth defects of the cuvA mutant we undertook a series of 343

additional experiments. We first examined cell shape and observed that the growth defect of M. 344

smegmatis ΔcuvA grown in cholesterol liquid MM was accompanied by altered cell morphology, 345

with cells that were shorter than wild type and appeared to have thickened, wider cell poles (Fig 346

4A). Measurement of the cholesterol-grown cells demonstrated that the difference in cell length 347

was highly significant (Fig. 4B). These cell length and morphology defects were fully 348

complemented by the wild type cuvAMs allele. When M. smegmatis ΔcuvA was grown on solid 349

MM plus cholesterol, cell morphology was also affected, with many cells showing asymmetric 350

bulging (Fig. 4C). This phenotype is very similar to the ΔyvcK morphology phenotype in B. 351

subtilis strains grown on gluconate, where the bifunctional PGN synthesis enzyme PBP1 is mis-352

localized (14). This M. smegmatis ΔcuvA phenotype is also very similar to the morphologic 353


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abnormalities seen in a PBP1 depletion strain of M. smegmatis, where PGN synthesis is directly 354

affected (30). 355

Because of the minimal growth of M. tuberculosis ΔcuvA on solid MM containing 356

cholesterol, there were too few cells to identify a morphologic phenotype of the ΔcuvA strain on 357

this medium. When ΔcuvA were grown to late stationary phase in liquid MM with 0.01% 358

cholesterol, however, we observed a bulging phenotype similar to that observed for M. 359

smegmatis (Fig. 4D). This phenotype was seen in 42% of ΔcuvA cells versus 11% of wild type 360

(P<0.0001 with over 200 cells analyzed per strain) and 12% of complemented cells. As was 361

seen for M. smegmatis, this M. tuberculosis phenotype was not seen for cells grown on MM plus 362

glucose. These morphologic phenotypes strongly suggest a defect in PGN synthesis or 363

structure in the ΔcuvA deletion strains. 364

CuvA localizes asymmetrically to the growing cell pole in mycobacteria 365

Peptidoglycan synthesis in mycobacteria and other actinomycetes is targeted to the 366

septum and asymmetrically to the growing cell pole (8, 31). The growth and morphology defects 367

we observed in the ΔcuvA strains, together with the data from B. subtilis indicating that YvcK 368

plays a role in regulating cell wall synthesis (14), led us to investigate the subcellular localization 369

of CuvA. We first determined that M. tuberculosis cuvA was a functional orthologue of the M. 370

smegmatis gene by showing that M. tuberculosis cuvA fully complements the cholesterol growth 371

defect of M. smegmatis ΔcuvA (Fig. S5). We then made a cuvAMt-gfp fusion and confirmed that 372

this construct also complemented the growth defect of the ΔcuvAMs strain in MM containing 373

0.01% cholesterol (Fig. S5). 374

Using this cuvAMt-gfp fusion, we found that CuvA-GFP strongly localizes to one pole of 375

most cells (Fig. 5), similar to the pattern we observed previously for Wag31 (DivIVA), an 376

essential protein that we showed to be required for polar localization of PGN synthesis in 377

mycobacteria (8). To determine the timing of CuvA localization and whether this protein is 378


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targeted to the old or new pole, we performed time-lapse live cell microscopy of M. smegmatis 379

expressing CuvA-GFP growing on nutrient agarose containing MM with 0.01% cholesterol 380

overlaid on microscope slides. As shown in the supplemental movie, we found that CuvA-GFP 381

localized to the old pole in most cells. We also observed that cell growth occurred predominantly 382

at one pole of the mycobacterial cell as we and others have shown previously (8, 32), and that 383

CuvA consistently localized to this growing pole. This localization of CuvA to the growing cell 384

pole occurred when cells were grown on medium containing either glucose or cholesterol as the 385

carbon source. Similar to these observations in M. smegmatis, CuvA also localized to one pole 386

in M. tuberculosis (Fig. 6). Though we were not able to perform time-lapse microscopy with M. 387

tuberculosis, observation of cells that appear to have recently undergone cell division indicates 388

that CuvA also localizes to the old, actively growing cell pole in this species. A non-389

phosphorylatable form of CuvA, in which Thr325 was replaced with Ala, also localized to the cell 390

pole in M. tuberculosis (not shown). 391

Our finding that CuvA localizes to the growing cell pole led us to predict that CuvA and 392

new PGN synthesis would co-localize. Fluorescent Vancomycin (Van-Alexa568), which binds to 393

the terminal D-Ala-D-Ala of newly synthesized peptidoglycan precursors, can be used to identify 394

sites of active PGN synthesis in bacteria (31). Using Van-Alexa568, we observed that most M. 395

smegmatis cells grown in glucose-containing medium were stained predominantly at one pole, 396

consistent with our previous observations (8), though some cells had a weaker signal from the 397

other pole and a few had approximately equal staining of both poles (Fig. 5). CuvA-GFP 398

consistently localized to the pole that showed strong Van-Alexa568 staining, indicating that 399

CuvA is targeted to sites of active PGN synthesis at the elongating cell pole. Surprisingly, 400

however, whereas asymmetry of PGN synthesis was seen in nearly all cells grown in glucose, in 401

cells grown on cholesterol, strong bipolar staining with fluorescent vancomycin was seen in the 402

majority of cells (Fig. 5). Despite this bipolar Vancomycin staining, CuvA consistently localized 403

to one pole in cholesterol-grown cells, indicating that CuvA is not absolutely required for 404


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localized PGN synthesis at the cell pole, but that it is consistently targeted to the old cell pole 405

when grown on either glucose or cholesterol. 406

Based on the findings in B. subtilis that the penicillin binding protein PBP1 is delocalized 407

in a yvck null mutant (14), we examined the localization of PBP1 in wild type M. smegmatis and 408

in the ΔcuvA strain. As previously reported (30), we found that a PBP1-RFP fusion protein 409

localizes to both cell poles and to the mid-cell in wild type M. smegmatis (Fig S6). In contrast to 410

the findings in B. subtilis, however, no differences were seen in PBP1-RFP localization in M. 411

smegmatis ΔcuvA compared to wild type, when grown on medium containing either glucose or 412

on cholesterol as the carbon source. Based on suppressor mutation results (see below) and its 413

identification as a probable substrate of PknB (6), we also examined the localization of PbpA. 414

As was observed with PBP1, the localization of PbpA was the same in wild type and the ΔcuvA 415

strain (data not shown). 416

417

The Ms-cuvA deletion strain is hyper-susceptible to β-lactam antibiotics and rifampin 418

Previous work had shown that M. smegmatis lacking the bifunctional penicillin binding 419

protein PBP1 is markedly more susceptible to several β-lactam antibiotics including ampicillin 420

and cephalothin (6 to >20 fold lower MIC), but shows little change in susceptibility to other 421

classes of antibiotics (33). In another study, a screen for M. smegmatis and M. tuberculosis 422

transposon mutants that are hypersusceptible to β-lactams identified several genes involved in 423

cell wall synthesis and cell division, including ponA2 (encoding PBP2), dapB and homologues of 424

DivIVA and DivIVC (34). Based on our data suggesting a role for CuvA in cell wall synthesis or 425

structure, we tested susceptibility of the M. tuberculosis and M. smegmatis deletion strains to 426

several antibiotics using the MABA assay. No significant differences in susceptibility of the M. 427

tuberculosis deletion strain compared to wild type were observed. Because of the high inoculum 428


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required for M. tuberculosis to grow in MM, however, there may be small differences in 429

susceptibility between strains that we could not detect with this assay (Table S1). 430

As shown in Table 1, however, we observed strikingly increased susceptibility of M. 431

smegmatis ΔcuvA to all four β-lactams tested when the strain was grown on cholesterol. For 432

ampicillin and the first generation cephalosporin cephalothin, the addition of the β-lactamase 433

inhibitor clavulanate was required to observe this increase in susceptibility, whereas for the third 434

generation cephalosporin cefotaxime and the carbapenem meropenem, which are less 435

susceptible to hydrolysis by β-lactamases, the increased susceptibility was evident without 436

addition of clavulanate (Table 1). This increased susceptibility phenotype was reversed in the 437

complemented strain. This hypersusceptibility was also reversed in each case by the addition of 438

0.2% glucose to the culture medium. The M. smegmatis mutant was not significantly more 439

susceptible to levofloxacin, isoniazid or gentamicin, none of which act on PGN synthesis. The 440

ΔcuvA strain was, however, significantly more susceptible to the RNA polymerase inhibitor 441

rifampin. While this finding raises the possibility of an effect of cuvA on RNA polymerase, an 442

indirect effect related to an altered cell wall in the mutant, such as increased uptake or 443

decreased efflux of rifampin, may account for this difference. 444

Suppressors of the CuvA cholesterol growth phenotype map to loci involved in cell wall 445

synthesis and transmembrane transport 446

Our phenotypic and localization data suggested that CuvA functions in utilization or 447

uptake of several carbon sources and is required for normal cell wall structure under nutrient-448

limited conditions. As another approach to gaining insight into the pathways in which CuvA 449

functions, we undertook a saturating transposon mutagenesis experiment to identify 450

suppressors of the M. tuberculosis ΔcuvA cholesterol growth defect. Genes in which two or 451

more insertions were obtained are shown in Table 2. A striking finding was the isolation of 452

several transductants with insertions in pbpA and rodA. These genes are involved in PGN 453


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synthesis and cell shape control and are part of the operon containing pknA and pknB, genes 454

encoding essential Ser/Thr kinases that regulate these processes (5, 6, 8, 9, 35). These 455

suppressors thus indicate a link between the ΔcuvA growth phenotype and cell wall synthesis 456

and cell morphology, and are reminiscent of the suppression of the growth and morphology 457

defects of the B. subtilis yvcK mutant by deletion of ponA, the gene encoding PBP1 (14). We 458

also obtained three independent insertions in the forward orientation at the beginning of 459

Rv2683, and one in the 5’ end of the Rv2683 coding sequence in the same orientation. The 460

location and orientation of these insertions, suggest that increased expression of this operon via 461

read-through expression from the transposon may be suppressing the ΔcuvA cholesterol growth 462

phenotype. Rv2683 is the first gene in a three gene operon that encodes a transporter that was 463

recently shown to be essential for M. tuberculosis growth on cholesterol (36). This result 464

suggests that increased expression of this transporter may be suppressing the ΔcuvA growth 465

defect. Two additional insertions disrupt the response regulator of an uncharacterized two-466

component system. 467

CuvA is required for virulence and persistence in a mouse infection model 468

In the context of data indicating that M. tuberculosis strains lacking the Mce4 cholesterol 469

transport system are attenuated during infection and a transposon mutagenesis study 470

suggesting that Rv1422 is required for bacterial replication in mice following intravenous 471

infection (12, 37), the in vitro growth defects of the ΔcuvA strains led us to compare the 472

virulence of M. tuberculosis ΔcuvA to the parental wild type H37Rv in mouse infection 473

experiments. Balb/C mice were infected by the aerosol route with wild type H37Rv, ΔcuvA and 474

ΔcuvA complemented strains. Examination of colony forming units (CFUs) present in lungs at 475

serial time points demonstrated that the wild type and ΔcuvA strains replicated at a similar rate 476

to day 14-28, after which the number of mutant bacteria declined (Fig. 7A). In contrast, the wild 477

type persisted at a stable level until day 84, after which a small decline was seen. At days 56, 478


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84 and 112 the difference between wild type and the cuvA strain was highly significant 479

(P<0.0001 to P<0.005). By the final time point at 112 days there was a 200-fold difference in 480

lung CFUs between these strains. The complemented strain showed an intermediate phenotype 481

with significantly more CFU in the lung than the ΔcuvA strain P<0.0001 to P<0.01), but fewer 482

CFU than the wild type (P<0.0001 to P<0.05). This persistence defect of the M. tuberculosis 483

cuvA mutant is similar to, but more pronounced than, the phenotype observed in an mce4 484

deletion strain, where decreased CFUs were seen at later stages of infection (28). In the spleen 485

the CFUs increased for all 3 strains to day 28, after which the wild type increased compared to 486

the mutant, though this phenotype was not well-complemented (Fig. S7). 487

Gross and microscopic pathology showed striking differences in the extent of disease in 488

the lungs from mice infected with the wild type and complemented strains compared to those 489

from ΔcuvA-infected mice. The lungs of wild type and complement-infected mice showed much 490

larger and more numerous tubercles, were significantly heavier (P<0.0001 to P<0.005 at days 491

54-112 for wild type vs. cuvA) and showed much more extensive cellular infiltrates on 492

histopathology (Fig. 7B-D). These phenotypes were well-complemented visually, and in the 493

case of lung weight statistically (P >0.05 at days 54-112 for wild type vs. complement). The 494

extent of disease in the spleen was also greater in wild type H37Rv-infected mice compared to 495

ΔcuvA-infected mice, though the difference was less striking (Fig. S7). 496

In separate time to death/severe morbidity experiments, mice were infected by aerosol, 497

four were sacrificed at day 1 to measure the inoculum (Fig. S7D) and the remaining mice were 498

monitored over time. This experiment also showed marked attenuation of the ΔcuvA strain. 50% 499

mortality for wild type H37Rv was reached at day 68, whereas by day 273 when the experiment 500

was ended, none of the mice infected with the ΔcuvA strain had died (P<0.0001) (Fig. 7E). 501

There was partial complementation, with 25% mortality of mice infected with the complemented 502

strain at day 84 (P=0.03 for ΔcuvA vs. complement). This incomplete complementation may 503


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have resulted from constitutive high level cuvA expression in the complemented strain, which 504

we found to be more than 15-fold higher than expression in the wild type. We did not observe 505

polar effects on the expression of genes adjacent to Rv1422 in the cuvA mutant (Fig. S8). 506

507

Discussion 508

In this work, we have provided an initial characterization of the M. tuberculosis gene of 509

unknown function, Rv1422, and it M. smegmatis orthologue MS_3080, which we have named 510

cuvA based on altered carbon source utilization in M. smegmatis and M. tuberculosis cuvA 511

deletion strains and decreased virulence phenotypes of the M. tuberculosis deletion mutant. 512

This protein has homologues across a broad range of diverse bacterial phyla and its amino acid 513

sequence suggests that CuvA is distantly related to the unidentified protein family UPF0052 514

(38). The one characterized member of this family is CofD (LPPG: 2-phospho-L-lactate 515

transferase), a protein present in archaea and some bacteria that has been shown to be 516

involved in coenzyme F420 biosynthesis in Methanocaldococcus jannaschii (39, 40). At the 517

level of primary amino acid sequence, however, CuvA is not significantly similar to CofD. 518

Another M. tuberculosis protein, FbiA (Rv3261), is highly similar to CofD and has been shown to 519

be required for F420 biosynthesis in M. bovis BCG (41), indicating that FbiA is the functional 520

orthologue of CofD and that CuvA likely has a different activity. 521

If it does not function in F420 biosynthesis, what is the role of CuvA? A B. subtilis strain 522

with a deletion in the cuvA orthologue yvcK was shown to have growth defects on several 523

carbon sources and to be defective in localization of PBP1 and cell wall synthesis. In this work 524

we have identified several phenotypes of M. tuberculosis and M. smegmatis cuvA deletion 525

mutants that suggest that the CuvA protein is required for utilization of nutrients and for normal 526

cell wall synthesis. In both species, the cuvA mutants show defects in utilization of several 527

carbon sources, including cholesterol, which is important for M. tuberculosis during infection in 528

vivo. In the case of M. smegmatis, growth defects were apparent when cells were grown in 529


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liquid MM or on agar MM. M. smegmatis ΔcuvA had growth defects on solid medium similar to 530

those seen in M. tuberculosis, with the exception that the M. smegmatis deletion strain grew 531

well on glucose but the M. tuberculosis strain did not. The basis of this difference in growth on 532

glucose will require further investigation, but might result from enhanced metabolic capacity 533

encoded in the much larger genome of the environmental organism M. smegmatis, or in the 534

more much more efficient uptake of glucose mediated via the MspA porin in M. smegmatis 535

compared to M. tuberculosis, which lacks this porin (42, 43). Growth defects of M. tuberculosis 536

ΔcuvA were not apparent in liquid medium. The very slow growth of M. tuberculosis in MM broth 537

and the requirement for a high inoculum for M. tuberculosis to grow in this medium may have 538

limited our ability to detect small differences. The mutant strains in both species had similar 539

morphologic abnormalities, with asymmetric bulging suggestive of a peptidoglycan defect. Also, 540

in both species CuvA localized to the growing cell pole, the site of peptidoglycan synthesis 541

required for cell elongation in actinomycetes including mycobacteria (8, 31). 542

A notable difference between the M. tuberculosis and M. smegmatis cuvA mutants was 543

the markedly increased susceptibility of the M. smegmatis, but not the M. tuberculosis strain, to 544

several cell-wall active antibiotics and to rifampin. Though differences in susceptibility of M. 545

tuberculosis ΔcuvA versus wild type to antibiotics may have been obscured by the poor growth 546

of M. tuberculosis in MM and the high inoculum needed to achieve growth, the magnitude of 547

differences seen in M. smegmatis, particularly for the cephalosporins, suggests that the 548

differences in this phenotype result from differences in the mycobacterial cell envelope in these 549

species. Taken together, the similarity of most phenotypes suggests that the function of CuvA is 550

conserved across these mycobacterial species. This interpretation is also supported by the 551

complementation of both the growth (Fig. S5) and antibiotic susceptibility (not shown) 552

phenotypes in M. smegmatis ΔcuvA by the cuvA gene of M. tuberculosis. 553


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The growth defect of the cuvA deletion strains was not specific to a single carbon 554

source. Growth rates of the cuvA mutants on each of the carbon sources tested, with the 555

exception of propionate, were comparable to or faster than on MM alone, in which asparagine is 556

the primary available energy source. Growth of M. smegmatis ΔcuvA on MM plus propionate, 557

however, was slower than on MM alone. Mycobacterial strains with defects in either the 558

glyoxalate cycle (icl1, icl2 double null mutant) or methylcitrate cycle (prpC, prpD double null 559

mutant) are unable to grow on cholesterol or propionate (44, 45). Though there is some 560

similarity in phenotypes of the ΔcuvA strains and the strains disrupted in glyoxalate or 561

methylcitrate pathways, there are important differences that suggest that CuvA does not 562

participate directly in these pathways. First, the M. tuberculosis strains with defects in the 563

glyoxylate or methylcitrate pathways show minimal growth on cholesterol or propionate in liquid 564

MM (17, 45, 46). In contrast the M. tuberculosis ΔcuvA strain did not have a significant defect in 565

liquid cholesterol medium and the M. smegmatis ΔcuvA strain grew more slowly but did achieve 566

sustained growth in MM plus cholesterol and MM plus propionate. Second, the M. smegmatis 567

ΔcuvA cholesterol growth defect was fully reversed by the addition of glucose, whereas the 568

growth defects of the icl1-2 and prpC-D M. tuberculosis strains were not reversed by glycerol. In 569

addition, the growth inhibitory effect of propionate on the M. smegmatis cuvA mutant was not 570

reversed by addition vitamin B12, in contrast to the prpC-D and icl1,2 mutants in which B12 571

addition allows cells to grow on propionate via utilization of the B12-dependent methylmalonyl 572

CoA pathway (17, 45). Thus, cuvA does not appear to be required for the glyoxylate or 573

methylcitrate pathways. 574

As noted above, our data indicate that the cuvA deletion mutant growth defect is not 575

specific and that CuvA is not an enzyme required for cholesterol catabolism. This inference is 576

consistent with a recent study using genetic approaches and metabolic profiling to map the 577

pathway of cholesterol catabolism in M. tuberculosis, in which Rv1422 was not identified as 578


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being required for any step in cholesterol degradation (17). These data, together with rescue of 579

the growth defect by addition of glucose also suggest that the growth defect is not the result of 580

accumulation of a toxic metabolite from catabolism of the several carbon sources on which the 581

ΔcuvA strain grew less well than wild type. Though a metabolic defect may explain the cuvA 582

growth phenotypes, these data, together with the genetic interaction of CuvA with both the mce-583

4 cholesterol transport locus and with the mce-1 locus that appears to be involved in turnover of 584

mycolic acids (11), suggest a possible alternative mechanism, i.e. that CuvA is required for 585

nutrient uptake into the mycobacterial cell. One mechanism for a broad effect on uptake of 586

many different nutrients would be alterations in the cell envelope in the ΔcuvA strains that affect 587

cell permeability or transporter functions. 588

Several of our results indicate that the mutant strains do have an altered cell envelope 589

and suggest that CuvA has a role in the structure and/or function of the mycobacterial cell wall. 590

First, in addition to decreased growth of the M. tuberculosis and M. smegmatis ΔcuvA mutants 591

on MM containing several carbon sources, these cells have an abnormal morphology, showing 592

shortened, bulging cells. This phenotype is strongly suggestive of an effect on cell wall integrity. 593

This altered morphology is highly similar to the previously described phenotype of an M. 594

smegmatis ponA depletion mutant, and similar to, though less severe than a wag31 depletion 595

mutant (8, 30); in both of these mutants, PGN synthesis is disrupted. The phenotype is also very 596

similar to the morphology of the B. subtilis yvcK deletion strain grown on gluconate, in which the 597

PGN biosynthetic enzyme PBP1 is mis-localized (8, 14, 30). The marked increase in 598

susceptibility of the M. smegmatis ΔcuvA strain to several β-lactam antibiotics, which parallels 599

the phenotype observed in a ponA depletion strain, also suggests that the cell wall is altered in 600

this strain. The localization of CuvA to the growing cell pole, the site of new PGN biosynthesis in 601

mycobacteria, is also consistent with a role for this gene in PGN synthesis, though our data 602

show that CuvA is not essential for this function. 603


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Suppressor mutagenesis can be a valuable means to gain insight into the pathway in 604

which a gene of unknown function is active. The suppressor mutagenesis results we obtained 605

provide further support a role for CuvA in cell wall synthesis and/or transport of nutrients. The 606

inactivating insertions present in pbpA and rodA that restore growth of M. tuberculosis ΔcuvA on 607

cholesterol likely affect the structure of the cell wall. This alteration may allow uptake of 608

cholesterol (and other nutrients) in the ΔcuvA strain in which cholesterol uptake is otherwise 609

impaired. In addition, for the insertions in Rv2683, their location at the 5’ end of the operon and 610

the orientation of all insertions in the direction of transcription of the operon suggest that these 611

may be gain of function mutations resulting from increased expression of genes in the operon 612

from the transposon promoter. Rv2683 is the first gene in a three gene operon that encodes a 613

transporter that was recently shown to be essential for M. tuberculosis growth on cholesterol 614

(36), suggesting that these insertions may enhance uptake of cholesterol and other compounds 615

into the mycobacterial cell. 616

The markedly decreased virulence of the cuvA deletion mutant in a mouse model of 617

tuberculosis suggests a critical role for this protein during infection. This attenuation was 618

especially pronounced in the lung, where both gross tubercle formation and microscopic 619

inflammation were markedly decreased in the ΔcuvA strain, and where CFU of the mutant 620

decreased significantly during the course of infection. Though M. tuberculosis appears to utilize 621

cholesterol throughout infection in mice (16), the small difference in lung CFU early during 622

infection and the progressive decline in CFU of the ΔcuvA strain that we observed during the 623

chronic phase parallels the persistence defect seen in an M. tuberculosis strain lacking the 624

Mce4 cholesterol transport system (2, 28). A second prior mouse infection study also showed 625

an mce4 deletion strain to be attenuated (37). In this study very small differences in lung CFU 626

were seen, along with decreased lung pathology and significantly increased survival of mice 627

infected with the mce4 strain. Compared to these studies, the decrease in lung CFU of the cuvA 628


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mutant in our infection experiment was more pronounced and the survival of the cuvA mutant 629

was more prolonged, even though we used a more susceptible mouse strain (Balb/C) than the 630

previous studies (c57BL/6). In the context of these prior data and the in vitro data presented 631

here, this result suggests that compromised utilization of cholesterol and other nutrients in the 632

ΔcuvA strain during infection likely contribute to the attenuation of the mutant. This interpretation 633

is also consistent with the genetic interaction study that first linked cuvA (Rv1422) to mce4 (28), 634

though other effects of the cuvA deletion, such as a defective cell wall, may also contribute to 635

decreased virulence. 636

The presence of CuvA homologues encoded in the genomes of a broad range of 637

bacteria suggests a highly conserved function for this protein. Though CuvA is required for 638

optimal nutrient utilization by mycobacteria, as for B. subtilis YvcK, the exact function of M. 639

tuberculosis CuvA remains to be defined at the molecular level. Our data, taken together with 640

the B. subtilis YvcK results, however, indicate a role for CuvA that links cell wall synthesis and 641

nutrient utilization. For M. tuberculosis, the severe attenuation of M. tuberculosis virulence and 642

persistence in the deletion strain suggest that CuvA may be a valuable target for development 643

of novel therapeutics to treat tuberculosis disease and/or latent tuberculosis infection. 644

645

646

647


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Acknowledgments 648

We thank members of the Husson laboratory for helpful discussions. This work was supported 649

by NIH grants AI059702 and AI099204 to RNH and by the NIH TARGET consortium contract 650

(N01-AI30036) to William Bishai, Johns Hopkins University. Microscopy resources were 651

provided by the Children’s Hospital Intellectual and Developmental Disabilities Research Center 652

(IDDRC) Cellular Imaging Core (NIH-P30-HD-1865) and New England Regional Center of 653

Excellence in Biodefense and Emerging Infectious Diseases live-cell imaging core (NIH grant 654

AI0571). All authors declare that they have no conflicts of interest.655


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46. Eoh H, Rhee KY. 2014. Methylcitrate cycle defines the bactericidal essentiality of 804 isocitrate lyase for survival of Mycobacterium tuberculosis on fatty acids. Proc 805 Natl Acad Sci U S A 111:4976-4981. 806

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810

Figure legends 811

Figure 1. Growth of M. tuberculosis strains in liquid and solid media. A. Wild type M. 812

tuberculosis H37Rv, ΔcuvAMt and complemented ΔcuvA (ΔcuvAMt/C) strains were grown in M-813

ADC-Tw liquid medium with 0.2% glucose, and OD600 was measured daily. B. Growth of the 814

same strains on agar plates containing MM plus 0.2% glucose or MM plus 0.01% cholesterol. 815

Serial 10-fold dilutions were spotted, incubated at 37o C and photographs were taken after one 816

month of incubation. In both panels, data shown are from one experiment that was repeated at 817

least twice with similar results. Glc, 0.2% glucose; Chol, 0.01% cholesterol. 818

819

Figure 2. Growth of M. tuberculosis strains on MM agar containing added carbon sources. Serial 820

10-fold dilutions of wild type M. tuberculosis H37Rv, ΔcuvAMt and complemented ΔcuvA 821

(ΔcuvAMt/C) strains were spotted on MM agar alone or containing the indicated compounds 822

added to a concentration of 0.01%, incubated at 37o C and photographs were taken after one 823

month of incubation. Data shown are from one experiment that was repeated at least twice with 824

similar results. Glc, glucose; Glyc, glycerol; Glcn, gluconate; Citr, citrate; Prop, propionate. 825

826

827

Figure 3. Growth of M. smegmatis strains on different carbon sources. M. smegmatis mc2-155 828

(WT); ΔcuvAMs, and complemented ΔcuvA (∆cuvAMs/C) were diluted to OD600=0.001 and grown 829

in liquid MM alone or with added carbon sources and analyzed using the MABA performed with 830

technical duplicates. Except for hiGlc, which contained 0.2% glucose, all compounds were 831

added to a concentration of 0.01%. The exponential growth phase of ΔcuvAMs was significantly 832

slower than wild type or ∆cuvAMs/C in MM alone (P<0.0001), MM plus cholesterol (P<0.0001), 833

MM plus glycerol (P<0.0001), MM plus citrate (P<0.005) and MM plus propionate ((P<0.0001). 834


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The growth rates were not significantly different (P> 0.05) when grown on 0.2% glucose, 0.01% 835

glucose or gluconate. Glc, glucose; Glyc, glycerol ; Citr, citrate;. Chol, cholesterol; Glcn, 836

gluconate; Prop, propionate. Error bars are +/- 1 SD. 837

838

Figure 4. Morphology of M. smegmatis and M. tuberculosis cells. A. M. smegmatis mc2-155 839

(Wild type); ΔcuvAMs, and complemented ΔcuvA (∆cuvAMs/C) were grown in MM plus 0.01% 840

cholesterol, harvested after 24h and examined by light microscopy. Bar = 1 μm. B. Length 841

distribution of M. smegmatis cells obtained after 24h of growth in MM plus 0.01% cholesterol, 842

determined by measuring over 200 cells of each strain, analyzed using imageJ software. The 843

horizontal line is mean cell length. C. Cells of each M. smegmatis strain were grown overnight 844

on agarose pads made with MM plus 0.01% cholesterol and photographed. D. Wild type M. 845

tuberculosis H37Rv, ΔcuvAMt and complemented ΔcuvA (ΔcuvAMt/C) strains were grown to late 846

stationary phase in MM liquid medium plus 0.01% cholesterol and examined by light microscopy 847

using a 100x oil immersion objective. Arrowheads in panels C and D indicate areas of abnormal 848

cell morphology. 849

850

851

Fig. 5. Co-localization of CuvA and nascent peptidoglycan synthesis in M. smegmatis. Wild 852

type M. smegmatis mc2-155 cells expressing GFP alone or GFP fused to CuvAMt were grown M-853

ADC-TW containing 0.2% glucose or MM containing 0.01% cholesterol. Fluorescent 854

Vancomycin labeled with Alexa-568 was used to bind to newly synthesized peptidoglycan. 855

Fluorescence and DIC microscopy images were obtained to determine the localization of 856

CuvAMt-GFP and nascent peptidoglycan synthesis. Bar = 1 μM. 857

858


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Fig. 6. Localization of CuvA in M. tuberculosis. An M. tuberculosis leucine/pantothenate 859

auxotroph strain (24) expressing CuvAMt-GFP or unfused GFP were grown on agarose pads 860

containing M-ADC medium with 0.2% glucose, and acetamide to induce expression of cuvAMt-861

gfp. Fluorescence and DIC microscopy images were obtained as described in the Methods 862

section after 24 hours induction to determine the localization of CuvAMt-GFP. Bar = 1 μM. 863

864

Fig. 7. Mouse infection experiments. Balb/C mice were infected by aerosol with wild type M. 865

tuberculosis wild type H37Rv, ΔcuvAMt or the ΔcuvA complemented strain (cuvAMt/C). In 866

separate experiments mice were monitored for morbidity/mortality or sacrificed at serial time 867

points for pathologic analysis and measurement of bacterial burden. A. CFUs in lungs from day 868

0 to day 112. Data are mean from four mice at each time point. Lungs from mice infected with 869

wild type had significantly more CFU in the lung than those infected with the ΔcuvA strain at 870

days 56, 84 and 112 (P<0.0001 to P<0.005). Mice infected with the complemented strain had 871

significantly more CFUs in the lung than those infected with the ΔcuvA strain at these time 872

points (P<0.0001 to P<0.01), but fewer CFUs than those infected with the wild type (P<0.0001 873

to P<0.05). Error bars = 1 S.D. B. Microscopic pathology of sections stained with hematoxylin 874

and eosin on day 84. C. Gross pathology of lungs on day 84. D. Lung weight from day 0 to day 875

112. Data are mean from four mice at each time point. Lungs of mice infected with the wild type 876

or complemented strain were significantly heavier than those infected with the ΔcuvA strain 877

(P<0.0001 to P<0.005 at days 54-112). Error bars = 1 S.D. E. Survival curve of mice infected 878

with the three strains of M. tuberculosis. Mortality was significantly lower in mice infected with 879

the ΔcuvA strain compared to those infected with wild type (P<0.0001) or the complemented 880

strain (P<0.05). Mortality in the complement-infected mice was also significantly lower than 881

those infected with wild type (P<0.0001). 882

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Table 1. Antibiotic Susceptibility of M. smegmatis strains 884 885

A Chol. Chol.+Glc Chol.+CLAV Chol.+Glc+CLAV

AMP

WT 375±137 250±0 63±0 52±16

ΔcuvA 229±51 250±0 5±2 31±0

Δ/C 375±137 250±0 31±0 47±38

CEF

WT >1000* >1000* >1000* >1000*

ΔcuvA 15.6±0.0 >1000* 7.3±0.8 >1000*

Δ/C >1000* >1000* >1000* >1000*

MER

WT 40.6±17.4

28.1±14.6

16.7±6.5 16.7±6.5

ΔcuvA 5.9±3.1 17.2±8.7 3.1±1.7 6.3±0.0

Δ/C 28.1±14.6

28.1±14.6

12.5±0.0 12.5±0.0

CEPH

WT >500* >500* >500* >500*

ΔcuvA >500* >500* 36.5±12.7 >500*

Δ/C >500* >500* >500* >500*

Data are minimal inhibitory concentration in μg/ml +/- 1 standard deviation. AMP, ampicillin; CEF, 886 cefotaxime; MER, meropenem; CEPH, cephalothin; RIF, rifampin; LEV, levofloxacin; INH, isoniazid; GEN, 887 gentamicin; CLAV, clavulanic acid. Chol, 0.01% cholesterol; Glc, 0.2% glucose. Shaded boxes indicate 888 results for ΔcuvA that are significantly different from wild type (P<0.05 and >2-fold difference). *Highest 889 concentration tested. 890

B Chol. Chol.+Glc

RIF

WT 26.7±10.3 16.3±5.2

ΔcuvA 2.3±1.9 15.6±6.2

Δ/C 17.5±5.0 16.7±5.2

LEV

WT 0.58±0.13 0.58±0.13

ΔcuvA 0.32±0.00 0.32±0.00

Δ/C 0.32±0.00 0.30±0.05

INH

WT >32* >32*

ΔcuvA >32* >32*

Δ/C >32* >32*

GEN

WT 0.08±0.02 0.08±0.02

ΔcuvA 0.08±0.03 0.08±0.02

Δ/C 0.08±0.02 0.07±0.03


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Table 2. Transposon insertions that suppress the cholesterol growth defect of M. 891 tuberculosis ΔcuvA 892

Genes in which two or more independent insertions were obtained are included in the Table. f and r in 893 parentheses indicate the forward or reverse orientation of the kanamycin marker in the transposon with 894 respect to the direction of transcription of the gene disrupted. The number preceding f or r indicates the 895 number of independent transposon insertions obtained at each site. 896

897

Gene disrupted

Insertion sites and number of insertions

Probable function of gene/operon

rodA +755 (2r), +1291 (1r) Cell shape/peptidoglycan synthesis, Ser/Thr phosphorylation

pbpA +142 (1r), +232 (1r), +250 (1f), +270 (1f), +541 (1f)

Peptidoglycan synthesis/cell shape, Ser/Thr phosphorylation

Rv2683 -1 (3f), +191 (1f) Conserved hypothetical regulatory protein/transmembrane transport

trcR +681 (1f), +732 (1r) Response regulator/TrcRS two component system


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Wild type cuvAMt cuvAMt/C

A

B

C

D


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Mycobacterial Gene cuvA Is Required for Optimal Nutrient Utilization and Virulence

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