Comparing rates of mycobacterial clearance in sputum smear-negative and smear-positive adults living with HIV.

BACKGROUND: Pulmonary tuberculosis (TB) in people living with HIV (PLH) frequently presents as sputum smear-negative. However, clinical trials of TB in adults often use smear-positive individuals to ensure measurable bacterial responses following initiation of treatment, thereby excluding HIV-infected patients from trials. METHODS: In this prospective case cohort study, 118 HIV-seropositive TB patients were assessed prior to initiation of standard four-drug TB therapy and at several time points through 35 days. Sputum bacillary load, as a marker of treatment response, was determined serially by: smear microscopy, Xpert MTB/RIF, liquid culture, and colony counts on agar medium. RESULTS: By all four measures, patients who were baseline smear-positive had higher bacterial loads than those presenting as smear-negative, until day 35. However, most smear-negative PLH had significant bacillary load at enrolment and their mycobacteria were cleared more rapidly than smear-positive patients. Smear-negative patients' decline in bacillary load, determined by colony counts, was linear to day 7 suggesting measurable bactericidal activity. Moreover, the decrease in bacterial counts was comparable to smear-positive individuals. Increasing cycle threshold values (Ct) on the Xpert assay in smear-positive patients to day 14 implied decreasing bacterial load. CONCLUSION: Our data suggest that smear-negative PLH can be included in clinical trials of novel treatment regimens as they contain sufficient viable bacteria, but allowances for late exclusions would have to be made in sample size estimations. We also show that increases in Ct in smear-positive patients to day 14 reflect treatment responses and the Xpert MTB/RIF assay could be used as biomarker for early treatment response.

Detection and Quantification of Differentially Culturable Tubercle Bacteria in Sputum from Patients with Tuberculosis.

RATIONALE: Recent studies suggest that baseline tuberculous sputum comprises a mixture of routinely culturable and differentially culturable tubercle bacteria (DCTB). The latter seems to be drug tolerant and dependent on resuscitation-promoting factors (Rpfs). OBJECTIVES: To further explore this, we assessed sputum from patients with tuberculosis for DCTB and studied the impact of exogenous culture filtrate (CF) supplementation ex vivo. METHODS: Sputum samples from adults with tuberculosis and HIV-1 and adults with no HIV-1 were used for most probable number (MPN) assays supplemented with CF and Rpf-deficient CF, to detect CF-dependent and Rpf-independent DCTB, respectively. MEASUREMENTS AND MAIN RESULTS: In 110 individuals, 19.1% harbored CF-dependent DCTB and no Rpf-independent DCTB. Furthermore, 11.8% yielded Rpf-independent DCTB with no CF-dependent DCTB. In addition, 53.6% displayed both CF-dependent and Rpf-independent DCTB, 1.8% carried CF-independent DCTB, and 13.6% had no DCTB. Sputum from individuals without HIV-1 yielded higher CF-supplemented MPN counts compared with counterparts with HIV-1. Furthermore, individuals with HIV-1 with CD4 counts greater than 200 cells/mm3 displayed higher CF-supplemented MPN counts compared with participants with HIV-1 with CD4 counts less than 200 cells/mm3. CF supplementation allowed for detection of mycobacteria in 34 patients with no culturable bacteria on solid media. Additionally, the use of CF enhanced detection of sputum smear-negative individuals. CONCLUSIONS: These observations demonstrate a novel Rpf-independent DCTB population in sputum and reveal that reduced host immunity is associated with lower prevalence of CF-responsive bacteria. Quantification of DCTB in standard TB diagnosis would be beneficial because these organisms provide a putative biomarker to monitor treatment response and risk of disease recurrence.

Activity of phosphino palladium(II) and platinum(II) complexes against HIV-1 and Mycobacterium tuberculosis.

Treatment of human immunodeficiency virus (HIV) is currently complicated by increased prevalence of co-infection with Mycobacterium tuberculosis. The development of drug candidates that offer the simultaneous management of HIV and tuberculosis (TB) would be of great benefit in the holistic treatment of HIV/AIDS, especially in sub-Saharan Africa which has the highest global prevalence of HIV-TB coinfection. Bis(diphenylphosphino)-2-pyridylpalladium(II) chloride (1), bis(diphenylphosphino)-2-pyridylplatinum(II) chloride (2), bis(diphenylphosphino)-2-ethylpyridylpalladium(II) chloride (3) and bis(diphenylphosphino)-2-ethylpyridylplatinum(II) (4) were investigated for the inhibition of HIV-1 through interactions with the viral protease. The complexes were subsequently assessed for biological potency against Mycobacterium tuberculosis H37Rv by determining the minimal inhibitory concentration (MIC) using broth microdilution. Complex (3) showed the most significant and competitive inhibition of HIV-1 protease (p = 0.014 at 100 µM). Further studies on its in vitro effects on whole virus showed reduced viral infectivity by over 80 % at 63 µM (p < 0.05). In addition, the complex inhibited the growth of Mycobacterium tuberculosis at an MIC of 5 µM and was non-toxic to host cells at all active concentrations (assessed by tetrazolium dye and real time cell electronic sensing). In vitro evidence is provided here for the possibility of utilizing a single metal-based compound for the treatment of HIV/AIDS and TB.

Development of primer-probe sets to rapidly distinguish single nucleotide polymorphisms in SARS-CoV-2 lineages.

Ongoing SARS-CoV-2 infections are driven by the emergence of various variants, with differential propensities to escape immune containment. Single nucleotide polymorphisms (SNPs) in the RNA genome result in altered protein structures and when these changes occur in the S-gene, encoding the spike protein, the ability of the virus to penetrate host cells to initiate an infection can be significantly altered. As a result, vaccine efficacy and prior immunity may be diminished, potentially leading to new waves of infection. Early detection of SARS-CoV-2 variants using a rapid and scalable approach will be paramount for continued monitoring of new infections. In this study, we developed minor groove-binding (MGB) probe-based qPCR assays targeted to specific SNPs in the S-gene, which are present in variants of concern (VOC), namely the E484K, N501Y, G446S and D405N mutations. A total of 95 archived SARS-CoV-2 positive clinical specimens collected in Johannesburg, South Africa between February 2021 and March 2022 were assessed using these qPCR assays. To independently confirm SNP detection, Sanger sequencing of the relevant region in the S-gene were performed. Where a PCR product could be generated and sequenced, qPCR assays were 100% concordant highlighting the robustness of the approach. These assays, and the approach described, offer the opportunity for easy detection and scaling of targeted detection of variant-defining SNPs in the clinical setting.

The performance of tongue swabs for detection of pulmonary tuberculosis.

Introduction: Oral and/or tongue swabs have demonstrated ability to detect Mycobacterium tuberculosis (Mtb) in adults with pulmonary tuberculosis (TB). Swabs provide useful alternative specimens for diagnosis of TB using molecular assays however, the diagnostic pickup by culture requires further improvement and development. Several studies identified the presence of differentially culturable tubercle bacilli (DCTB) populations in a variety of clinical specimens. These organisms do not grow in routine laboratory media and require growth factors in the form of culture filtrate (CF) from logarithmic phase cultures of Mtb H37Rv. Methods: Herein, we compared the diagnostic performance of sputum and tongue swabs using Mycobacterial Growth Indicator Tube (MGIT) assays, Auramine smear, GeneXpert and DCTB assays supplemented with or without CF. Results: From 89 eligible participants, 83 (93%), 66 (74%) and 79 (89%) were sputum positive by MGIT, smear and GeneXpert, respectively. The corresponding tongue swabs displayed a lower sensitivity with 39 (44%), 2 (2.0%) and 18 (20%) participants respectively for the same tests. We aimed to improve the diagnostic yield by utilizing DCTB assays. Sputum samples were associated with a higher positivity rate for CF-augmented DCTB at 82/89 (92%) relative to tongue swabs at 36/89 (40%). Similarly, sputum samples had a higher positivity rate for DCTB populations that were CF-independent at 64/89 (72%) relative to tongue swabs at 26/89 (29%). DCTB positivity increased significantly, relative to MGIT culture, for tongue swabs taken from HIV-positive participants. We next tested whether the use of an alternative smear stain, DMN-Trehalose, would improve diagnostic yield but noted no substantial increase. Discussion: Collectively, our data show that while tongue swabs yield lower bacterial numbers for diagnostic testing, the use of growth supplementation may improve detection of TB particularly in HIV-positive people but this requires further interrogation in larger studies.

Survival and detection of SARS-CoV-2 variants on dry swabs post storage.

COVID-19 has resulted in nearly 598 million infections and over 6.46 million deaths since the start of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic in 2019. The rapid onset of the pandemic, combined with the emergence of viral variants, crippled many health systems particularly from the perspective of coping with massive diagnostic loads. Shortages of diagnostic kits and capacity forced laboratories to store clinical samples resulting in huge backlogs, the effects of this on diagnostic pickup have not been fully understood. Herein, we investigated the impact of storing SARS-CoV-2 inoculated dry swabs on the detection and viability of four viral strains over a period of 7 days. Viral load, as detected by qRT-PCR, displayed no significant degradation during this time for all viral loads tested. In contrast, there was a ca. 2 log reduction in viral viability as measured by the tissue culture infectious dose (TCID) assay, with 1-3 log viable virus detected on dry swabs after 7 days. When swabs were coated with 102 viral copies of the Omicron variant, no viable virus was detected after 24 hours following storage at 4°C or room temperature. However there was no loss of PCR signal over 7 days. All four strains showed comparable growth kinetics and survival when cultured in Vero E6 cells. Our data provide information on the viability of SARS-CoV-2 on stored swabs in a clinical setting with important implications for diagnostic pickup and laboratory processing protocols. Survival after 7 days of SARS-CoV-2 strains on swabs with high viral loads may impact public health and biosafety practices in diagnostic laboratories.

The detection of mixed tuberculosis infections using culture filtrate and resuscitation promoting factor deficient filtrate.

Tuberculosis (TB) infected individuals harbor a heterogenous population of differentially culturable tubercle bacilli (DCTB). Herein, we describe how DCTB assays using culture filtrate either containing or deficient in resuscitation promoting factors can uncover mixed infections. We demonstrate that Mycobacterium tuberculosis (Mtb) strain genotypes can be separated in DCTB assays based on their selective requirement for growth stimulatory factors. Beijing mixed infections appear to be associated with a higher bacterial load and reduced reliance on growth stimulatory factors. These data have important implications for identifying mixed infections and hetero-resistance, which in turn can affect selection of treatment regimen and establishment of transmission links.

Detection of differentially culturable tubercle bacteria in sputum from drug-resistant tuberculosis patients.

Several studies described the presence of non-replicating, drug-tolerant differentially culturable tubercle bacteria (DCTB) in sputum from patients with active tuberculosis (TB). These organisms are unable to form colonies on agar but can be recovered in liquid media supplemented with culture filtrate as a source of growth factors. Herein, we undertook to investigate the response of DCTB during the treatment of individuals with drug-resistant TB. A cohort of 100 participants diagnosed with rifampicin-resistant TB were enrolled and prospectively followed to monitor response to therapy using routine culture and limiting dilution assays, supplemented with culture filtrate (CF) to quantify DCTB. Fifteen participants were excluded due to contamination, and of the remaining 85 participants, 29, 49, and 7 were infected with rifampicin mono-resistant (RMR), multidrug-resistant (MDR), or extremely drug-resistant (XDR) TB, respectively. Analysis of baseline sputum demonstrated that CF supplementation of limiting dilution assays detected notable amounts of DCTB. Prevalence of DCTB was not influenced by smear status or mycobacterial growth indicator tube time to positivity. CF devoid of resuscitation promoting factors (Rpfs) yielded a greater amount of DCTB in sputum from participants with MDR-TB compared with those with RMR-TB. A similar effect was noted in DCTB assays without CF supplementation, suggesting that CF is dispensable for the detection of DCTB from drug-resistant strains. The HIV status of participants, and CD4 count, did not affect the amount of DCTB recovered. During treatment with second-line drug regimens, the probability of detecting DCTB from sputum specimens in liquid media with or without CF was higher compared with colony forming units, with DCTB detected up to 16 weeks post treatment. Collectively, these data point to differences in the ability of drug-resistant strains to respond to CF and Rpfs. Our findings demonstrate the possible utility of DCTB assays to diagnose and monitor treatment response for drug-resistant TB, particularly in immune compromised individuals with low CD4 counts.

Differentially culturable tubercle bacteria as a measure of tuberculosis treatment response.

Introduction: Routine efficacy assessments of new tuberculosis (TB) treatments include quantitative solid culture or routine liquid culture, which likely miss quantification of drug tolerant bacteria. To improve these assessments, comparative analyses using additional measures such as quantification of differentially culturable tubercle bacteria (DCTB) are required. Essential for enabling this is a comparative measure of TB treatment responses using routine solid and liquid culture with liquid limiting dilutions (LLDs) that detect DCTB in sputum. Methods: We recruited treatment-naïve TB patients, with and without HIV-infection, and serially quantified their sputum for DCTB over the course of treatment. Results: Serial sputum sampling in 73 individuals during their first 14 days of treatment demonstrated that clearance of DCTB was slower compared to routine solid culture. Treatment response appeared to be characterized by four patterns: (1) Classic bi-phasic bacterial clearance; (2) early non-responders with slower clearance; (3) paradoxical worsening with an increase in bacterial count upon treatment initiation; and (4) non-responders with no change in bacterial load. During treatment, LLDs displayed greater bacterial yield when compared with quantitative solid culture. Upon treatment completion, 74% [46/62] of specimens displayed residual DCTB and within this group, two recurrences were diagnosed. Residual DCTB upon treatment completion was associated with a higher proportion of MGIT culture, GeneXpert, and smear positivity at two months post treatment. No recurrences occurred in the group without residual DCTB. Discussion: These data indicate that DCTB assays detect distinct subpopulations of organisms in sputum that are missed by routine solid and liquid culture, and offer important alternatives for efficacy assessments of new TB treatments. The residual DCTB observed upon treatment completion suggests that TB treatment does not always eliminate all bacterial populations, a finding that should be investigated in larger cohorts.

Detection of differentially culturable tubercle bacteria in sputum using mycobacterial culture filtrates.

Rapid detection of tuberculosis (TB) infection is paramount to curb further transmission. The gold standard for this remains mycobacterial culture, however emerging evidence confirms the presence of differentially culturable tubercle bacteria (DCTB) in clinical specimens. These bacteria do not grow under standard culture conditions and require the presence of culture filtrate (CF), from axenic cultures of Mycobacterium tuberculosis (Mtb), to emerge. It has been hypothesized that molecules such as resuscitation promoting factors (Rpfs), fatty acids and cyclic-AMP (cAMP) present in CF are responsible for the growth stimulatory activity. Herein, we tested the ability of CF from the non-pathogenic bacterium Mycobacterium smegmatis (Msm) to stimulate the growth of DCTB, as this organism provides a more tractable source of CF. We also interrogated the role of Mtb Rpfs in stimulation of DCTB by creating recombinant strains of Msm that express Mtb rpf genes in various combinations. CF derived from this panel of strains was tested on sputum from individuals with drug susceptible TB prior to treatment. CF from wild type Msm did not enable detection of DCTB in a manner akin to Mtb CF preparations and whilst the addition of RpfABMtb and RpfABCDEMtb to an Msm mutant devoid of its native rpfs did improve detection of DCTB compared to the no CF control, it was not statistically different to the empty vector control. To further investigate the role of Rpfs, we compared the growth stimulatory activity of CF from Mtb, with and without Rpfs and found these to be equivalent. Next, we tested chemically diverse fatty acids and cAMP for growth stimulation and whilst some selective stimulatory effect was observed, this was not significantly higher than the media control and not comparable to CF. Together, these data indicate that the growth stimulatory effect observed with Mtb CF is most likely the result of a combination of factors. Future work aimed at identifying the nature of these growth stimulatory molecules may facilitate improvement of culture-based diagnostics for TB.

Role of the DinB homologs Rv1537 and Rv3056 in Mycobacterium tuberculosis.

The environment encountered by Mycobacterium tuberculosis during infection is genotoxic. Most bacteria tolerate DNA damage by engaging specialized DNA polymerases that catalyze translesion synthesis (TLS) across sites of damage. M. tuberculosis possesses two putative members of the DinB class of Y-family DNA polymerases, DinB1 (Rv1537) and DinB2 (Rv3056); however, their role in damage tolerance, mutagenesis, and survival is unknown. Here, both dinB1 and dinB2 are shown to be expressed in vitro in a growth phase-dependent manner, with dinB2 levels 12- to 40-fold higher than those of dinB1. Yeast two-hybrid analyses revealed that DinB1, but not DinB2, interacts with the beta-clamp, consistent with its canonical C-terminal beta-binding motif. However, knockout of dinB1, dinB2, or both had no effect on the susceptibility of M. tuberculosis to compounds that form N(2)-dG adducts and alkylating agents. Similarly, deletion of these genes individually or in combination did not affect the rate of spontaneous mutation to rifampin resistance or the spectrum of resistance-conferring rpoB mutations and had no impact on growth or survival in human or mouse macrophages or in mice. Moreover, neither gene conferred a mutator phenotype when expressed ectopically in Mycobacterium smegmatis. The lack of the effect of altering the complements or expression levels of dinB1 and/or dinB2 under conditions predicted to be phenotypically revealing suggests that the DinB homologs from M. tuberculosis do not behave like their counterparts from other organisms.

Depletion of resuscitation-promoting factors has limited impact on the drug susceptibility of Mycobacterium tuberculosis.

OBJECTIVES: Mycobacterium tuberculosis has five homologues of the muralytic resuscitation-promoting factor (Rpf), which are collectively dispensable for growth in vitro, but are required for resuscitation from a non-culturable state and for virulence in a mouse infection model. Our study was aimed at assessing the drug susceptibility of mutants lacking all five rpf genes. METHODS: Drug susceptibility was determined in liquid medium using the broth microdilution method. RESULTS: Strains of M. tuberculosis that lack all five rpf-like genes display a 4-8-fold increase in susceptibility to vancomycin and erythromycin, but show no significant difference in susceptibility to other antibiotics tested, including first- and second-line antitubercular agents. CONCLUSIONS: These results suggest that the possible alterations in the peptidoglycan structure and/or turnover caused by Rpf deficiency have a limited impact on the drug susceptibility of M. tuberculosis.

The carbon starvation-inducible genes Rv2557 and Rv2558 of Mycobacterium tuberculosis are not required for long-term survival under carbon starvation and for virulence in SCID mice.

Mycobacterium tuberculosis genes Rv2557 and Rv2558 have no known function. However, proteome, transcriptome and in situ hybridization studies have shown that these genes are significantly upregulated under carbon-starved conditions and in human granulomas, suggesting that they may play a role in persistence. Single and double deletion mutants of M. tuberculosis H37Rv in Rv2557 and/or Rv2558 were generated to explore their individual and/or collective role(s) in growth and survival. The mutants were assessed for growth and survival in vitro under normal and nutrient-deprived conditions and for virulence in the SCID mouse model. Although highly induced by carbon starvation, loss of Rv2557 and/or Rv2558 affected neither the long-term survival of M. tuberculosis under carbon-starved conditions in vitro, nor its virulence in SCID mice.

A combinatorial role for MutY and Fpg DNA glycosylases in mutation avoidance in Mycobacterium smegmatis.

Hydroxyl radical (OH) among reactive oxygen species cause damage to nucleobases with thymine being the most susceptible, whilst in contrast, the singlet oxygen ((1)02) targets only guanine bases. The high GC content of mycobacterial genomes predisposes these organisms to oxidative damage of guanine. The exposure of cellular DNA to OH and one-electron oxidants results in the formation of two main degradation products, the pro-mutagenic 8-oxo-7,8-dihydroguanine (8-oxoGua) and the cytotoxic 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua). These lesions are repaired through the base excision repair (BER) pathway and we previously, demonstrated a combinatorial role for the mycobacterial Endonuclease III (Nth) and the Nei family of DNA glycosylases in mutagenesis. In addition, the formamidopyrimidine (Fpg/MutM) and MutY DNA glycosylases have also been implicated in mutation avoidance and BER in mycobacteria. In this study, we further investigate the combined role of MutY and the Fpg/Nei DNA glycosylases in Mycobacterium smegmatis and demonstrate that deletion of mutY resulted in enhanced sensitivity to oxidative stress, an effect which was not exacerbated in Δfpg1 Δfpg2 or Δnei1 Δnei2 double mutant backgrounds. However, combinatorial loss of the mutY, fpg1 and fpg2 genes resulted in a significant increase in mutation rates suggesting interplay between these enzymes. Consistent with this, there was a significant increase in C → A mutations with a corresponding change in cell morphology of rifampicin resistant mutants in the Δfpg1 Δfpg2 ΔmutY deletion mutant. In contrast, deletion of mutY together with the nei homologues did not result in any growth/survival defects or changes in mutation rates. Taken together these data indicate that the mycobacterial mutY, in combination with the Fpg DNA N-glycosylases, plays an important role in controlling mutagenesis under oxidative stress.

The contribution of Nth and Nei DNA glycosylases to mutagenesis in Mycobacterium smegmatis.

The increased prevalence of drug resistant strains of Mycobacterium tuberculosis (Mtb) indicates that significant mutagenesis occurs during tuberculosis disease in humans. DNA damage by host-derived reactive oxygen/nitrogen species is hypothesized to be critical for the mutagenic process in Mtb thus, highlighting an important role for DNA repair enzymes in maintenance of genome fidelity. Formamidopyrimidine (Fpg/MutM/Fapy) and EndonucleaseVIII (Nei) constitute the Fpg/Nei family of DNA glycosylases and together with EndonucleaseIII (Nth) are central to the base excision repair pathway in bacteria. In this study we assess the contribution of Nei and Nth DNA repair enzymes in Mycobacterium smegmatis (Msm), which retains a single nth homologue and duplications of the Fpg (fpg1 and fpg2) and Nei (nei1 and nei2) homologues. Using an Escherichia coli nth deletion mutant, we confirm the functionality of the mycobacterial nth gene in the base excision repair pathway. Msm mutants lacking nei1, nei2 and nth individually or in combination did not display aberrant growth in broth culture. Deletion of nth individually results in increased UV-induced mutagenesis and combinatorial deletion with the nei homologues results in reduced survival under oxidative stress conditions and an increase in spontaneous mutagenesis to rifampicin. Deletion of nth together with the fpg homolgues did not result in any growth/survival defects or changes in mutation rate. Furthermore, no differential emergence of the common rifampicin resistance conferring genotypes were noted. Collectively, these data confirm a role for Nth in base excision repair in mycobacteria and further highlight a novel interplay between the Nth and Nei homologues in spontaneous mutagenesis.

VapC toxins from Mycobacterium tuberculosis are ribonucleases that differentially inhibit growth and are neutralized by cognate VapB antitoxins.

The chromosome of Mycobacterium tuberculosis (Mtb) encodes forty seven toxin-antitoxin modules belonging to the VapBC family. The role of these modules in the physiology of Mtb and the function(s) served by their expansion are unknown. We investigated ten vapBC modules from Mtb and the single vapBC from M. smegmatis. Of the Mtb vapCs assessed, only Rv0549c, Rv0595c, Rv2549c and Rv2829c were toxic when expressed from a tetracycline-regulated promoter in M. smegmatis. The same genes displayed toxicity when conditionally expressed in Mtb. Toxicity of Rv2549c in M. smegmatis correlated with the level of protein expressed, suggesting that the VapC level must exceed a threshold for toxicity to be observed. In addition, the level of Rv2456 protein induced in M. smegmatis was markedly lower than Rv2549c, which may account for the lack of toxicity of this and other VapCs scored as 'non-toxic'. The growth inhibitory effects of toxic VapCs were neutralized by expression of the cognate VapB as part of a vapBC operon or from a different chromosomal locus, while that of non-cognate antitoxins did not. These results demonstrated a specificity of interaction between VapCs and their cognate VapBs, a finding corroborated by yeast two-hybrid analyses. Deletion of selected vapC or vapBC genes did not affect mycobacterial growth in vitro, but rendered the organisms more susceptible to growth inhibition following toxic VapC expression. However, toxicity of 'non-toxic' VapCs was not unveiled in deletion mutant strains, even when the mutation eliminated the corresponding cognate VapB, presumably due to insufficient levels of VapC protein. Together with the ribonuclease (RNase) activity demonstrated for Rv0065 and Rv0617--VapC proteins with similarity to Rv0549c and Rv3320c, respectively--these results suggest that the VapBC family potentially provides an abundant source of RNase activity in Mtb, which may profoundly impact the physiology of the organism.

The resuscitation-promoting factors of Mycobacterium tuberculosis are required for virulence and resuscitation from dormancy but are collectively dispensable for growth in vitro.

Mycobacterium tuberculosis contains five resuscitation-promoting factor (Rpf)-like proteins, RpfA-E, that are implicated in resuscitation of this organism from dormancy via a mechanism involving hydrolysis of the peptidoglycan by Rpfs and partnering proteins. In this study, the rpfA-E genes were shown to be collectively dispensable for growth of M. tuberculosis in broth culture. The defect in resuscitation of multiple mutants from a 'non-culturable' state induced by starvation under anoxia was reversed by genetic complementation or addition of culture filtrate from wild-type organisms confirming that the phenotype was associated with rpf-like gene loss and that the 'non-culturable' cells of the mutant strains were viable. Other phenotypes uncovered by sequential deletion mutagenesis revealed a functional differentiation within this protein family. The quintuple mutant and its parent that retained only rpfD displayed delayed colony formation and hypersensitivity to detergent, effects not observed for mutants retaining only rpfE or rpfB. Furthermore, mutants retaining rpfD or rpfE were highly attenuated for growth in mice with the latter persisting better than the former in late-stage infection. In conjunction, these results are indicative of a hierarchy in terms of function and/or potency with the Rpf family, with RpfB and RpfE ranking above RpfD.

Production of mutants in amino acid biosynthesis genes of Mycobacterium tuberculosis by homologous recombination.

The ability to generate mutants of Mycobacterium tuberculosis will be important if we are to understand the biology of this major pathogen. However, allelic replacement methods have only recently achieved success. We have developed a reproducible method for generating defined mutants of M. tuberculosis using homologous recombination. The transforming DNA was used following pre-treatment either with UV light or alkali denaturation in order to stimulate homologous recombination and abolish illegitimate recombination. Suicide vectors carrying one of nine amino acid biosynthesis genes were electroporated into M. tuberculosis, and homologous recombinants were obtained in all nine genes; eight resulted from single-crossover events (SCOs) and one from a double-crossover event (DCO) (in the metB gene). The remaining colonies were spontaneous hygromycin-resistant mutants; no products of illegitimate recombination were observed. To more efficiently distinguish spontaneous mutants, the lacZ gene was cloned into five vectors (two containing genes not previously tested), and the transformations were repeated. SCO mutants were identified by screening for blue colonies on indicator plates. White transformants were tested for auxotrophy and trpD, hisD and proC auxotrophic mutants were obtained. Only blue SCOs were obtained for argF and glnE. Thus, using this methodology we have obtained homologous recombination in 11 genes, and DCOs in 4 genes, showing that it is possible to generate targeted mutants in a reproducible way.

Construction and phenotypic characterization of an auxotrophic mutant of Mycobacterium tuberculosis defective in L-arginine biosynthesis.

A mutant of Mycobacterium tuberculosis defective in the metabolism of L-arginine was constructed by allelic exchange mutagenesis. The argF mutant strain required exogenous L-arginine for growth in vitro, and in the presence of 0.96 mM L-arginine, it achieved a growth rate and cell density in stationary phase comparable to those of the wild type. The mutant strain was also able to grow in the presence of high concentrations of argininosuccinate, but its auxotrophic phenotype could not be rescued by L-citrulline, suggesting that the DeltaargF::hyg mutation exerted a polar effect on the downstream argG gene but not on argH. The mutant strain displayed reduced virulence in immunodeficient SCID mice and was highly attenuated in immunocompetent DBA/2 mice, suggesting that L-arginine availability is restricted in vivo.

The Mycobacterium tuberculosis protein serine/threonine kinase PknG is linked to cellular glutamate/glutamine levels and is important for growth in vivo.

The function of the Mycobacterium tuberculosis eukaryotic-like protein serine/threonine kinase PknG was investigated by gene knock-out and by expression and biochemical analysis. The pknG gene (Rv0410c), when cloned and expressed in Escherichia coli, encodes a functional kinase. An in vitro kinase assay of the recombinant protein demonstrated that PknG can autophosphorylate its kinase domain as well as its 30 kDa C-terminal portion, which contains a tetratricopeptide (TPR) structural signalling motif. Western analysis revealed that PknG is located in the cytosol as well as in mycobacterial membrane. The pknG gene was inactivated by allelic exchange in M. tuberculosis. The resulting mutant strain causes delayed mortality in SCID mice and displays decreased viability both in vitro and upon infection of BALB/c mice. The reduced growth of the mutant was more pronounced in the stationary phase of the mycobacterial growth cycle and when grown in nutrient-depleted media. The PknG-deficient mutant accumulates glutamate and glutamine. The cellular levels of these two amino acids reached approximately threefold of their parental strain levels. Higher cellular levels of the amine sugar-containing molecules, GlcN-Ins and mycothiol, which are derived from glutamate, were detected in the DeltapknG mutant. De novo glutamine synthesis was shown to be reduced by 50%. This is consistent with current knowledge suggesting that glutamine synthesis is regulated by glutamate and glutamine levels. These data support our hypothesis that PknG mediates the transfer of signals sensing nutritional stress in M. tuberculosis and translates them into metabolic adaptation.