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.

Production and Performance Assessment of a Severe Acute Respiratory Syndrome Coronavirus 2 Biomimetic in a Verification Program for Pandemic Readiness.

During the early stages of the 2019 coronavirus disease (COVID-19) pandemic in South Africa, one of many challenges included availability of control material for laboratory proficiency testing programs. Proficiency testing control material using live severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or RNA extracted from cell culture was either biohazardous or costly, particularly in resource-limited settings. This study reports the development and application of a noninfectious SARS-CoV-2 biomimetic Mycobacterium smegmatis strain that mimics a positive result in the GeneXpert SARS-CoV-2 Xpert Xpress cartridge. Nucleotide sequences located in genes encoding the RNA-dependent RNA polymerase, the nucleocapsid, and the envelope proteins were used. The resulting biomimetic strain was prepared as a positive proficiency testing control and distributed in South Africa for verification of laboratories before their testing of clinical specimens. Between April and December 2020, a total of 151 GeneXpert instruments with 2532 modules were verified to bring COVID-19 mass testing in South Africa online. An average concordance of 98.6% was noted in the entire laboratory network, allowing identification of false-positive/false-negative results and instrument errors. This noninfectious, easily scalable proficiency testing control material became available within 2 months after the start of the pandemic in South Africa and represents a useful approach to consider for other diseases and future pandemics.

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 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.

ß-lactam resistance: The role of low molecular weight penicillin binding proteins, ß-lactamases and ld-transpeptidases in bacteria associated with respiratory tract infections.

Disruption of peptidoglycan (PG) biosynthesis in the bacterial cell wall by ß-lactam antibiotics has transformed therapeutic options for bacterial infections. These antibiotics target the transpeptidase domains in penicillin binding proteins (PBPs), which can be classified into high and low molecular weight (LMW) counterparts. While the essentiality of the former has been extensively demonstrated, the physiological roles of LMW PBPs remain poorly understood. Herein, we review the function of LMW PBPs, ß-lactamases and ld-transpeptidases (Ldts) in pathogens associated with respiratory tract infections. More specifically, we explore their roles in mediating ß-lactam resistance. Using a comparative genomics approach, we identified a high degree of genetic redundancy for LMW PBPs which retain the motifs, SxxN, SxN and KTG required for catalytic activity. Differences in domain architecture suggest distinct physiological roles, possibly related to bacterial cell cycle and/or adaptation to various environmental conditions. Many of the LMW PBPs play an important role in ß-lactam resistance either through mutation or variation in abundance. In all of the bacterial genomes assessed, at least one ß-lactamase homologue is present, suggesting that enzymatic degradation of ß-lactams is a highly conserved resistance mechanism. Furthermore, the presence of Ldt homologues in the majority of species surveyed suggests that alternative PG crosslinking may further mediate ß-lactam drug resistance. A deeper understanding of the interplay between these different mechanisms of ß-lactam resistance will provide a framework for new therapeutics, which are urgently required given the rapid emergence of antimicrobial resistance. © 2018 IUBMB Life, 70(9):855-868, 2018.

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.

Essential roles for imuA'- and imuB-encoded accessory factors in DnaE2-dependent mutagenesis in Mycobacterium tuberculosis.

In Mycobacterium tuberculosis (Mtb), damage-induced mutagenesis is dependent on the C-family DNA polymerase, DnaE2. Included with dnaE2 in the Mtb SOS regulon is a putative operon comprising Rv3395c, which encodes a protein of unknown function restricted primarily to actinomycetes, and Rv3394c, which is predicted to encode a Y-family DNA polymerase. These genes were previously identified as components of an imuA-imuB-dnaE2-type mutagenic cassette widespread among bacterial genomes. Here, we confirm that Rv3395c (designated imuA') and Rv3394c (imuB) are individually essential for induced mutagenesis and damage tolerance. Yeast two-hybrid analyses indicate that ImuB interacts with both ImuA' and DnaE2, as well as with the beta-clamp. Moreover, disruption of the ImuB-beta clamp interaction significantly reduces induced mutagenesis and damage tolerance, phenocopying imuA', imuB, and dnaE2 gene deletion mutants. Despite retaining structural features characteristic of Y-family members, ImuB homologs lack conserved active-site amino acids required for polymerase activity. In contrast, replacement of DnaE2 catalytic residues reproduces the dnaE2 gene deletion phenotype, strongly implying a direct role for the alpha-subunit in mutagenic lesion bypass. These data implicate differential protein interactions in specialist polymerase function and identify the split imuA'-imuB/dnaE2 cassette as a compelling target for compounds designed to limit mutagenesis in a pathogen increasingly associated with drug resistance.

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.

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.

In vitro analysis of rates and spectra of mutations in a polymorphic region of the Rv0746 PE_PGRS gene of Mycobacterium tuberculosis.

An assay modeled on a known polymorphism in the PE_PGRS9 gene of Mycobacterium tuberculosis was designed to assess the mutability of a sequence containing interspersed PGRS repeats. Application of the assay in Mycobacterium smegmatis revealed sequence plasticity: in addition to recapitulating the mutation on which it was based, other mutations likely mediated by replication slippage between PGRS repeats were detected. However, the mutation rates argued against marked hypermutability of such sequences in mycobacteria.

TB tools to tell the tale-molecular genetic methods for mycobacterial research.

In spite of the availability of drugs and a vaccine, tuberculosis--one of man's medical nemeses--remains a formidable public health problem, particularly in the developing world. The persistent nature of the tubercle bacillus, with one third of the world's population is estimated to be infected, combined with the emergence of multi drug-resistant strains and the exquisite susceptibility of HIV-positive individuals, has underscored the urgent need for in-depth study of the biology of Mycobacterium tuberculosis address the resurgence of TB. In aiming to understand the mechanisms by which mycobacteria react to their immediate environments, molecular genetic tools have been developed from naturally occurring genetic elements. These include protein expressing genes, and episomal and integrating elements, which have been derived mainly from prokaryotic but also from eukaryotic organisms. Molecular genetic tools that had been established as routine procedures in other prokaryotic genera were thus mimicked. Knowledge of the underlying mechanisms greatly expedited the harnessing of these elements for mycobacteriological research and has brought us to a point where these molecular genetic tools are now employed routinely in laboratories worldwide.

DNA alkylation damage as a sensor of nitrosative stress in Mycobacterium tuberculosis.

One of the cellular consequences of nitrosative stress is alkylation damage to DNA. To assess whether nitrosative stress is registered on the genome of Mycobacterium tuberculosis, mutants lacking an alkylation damage repair and reversal operon were constructed. Although hypersensitive to the genotoxic effects of N-methyl-N'-nitro-N-nitrosoguanidine in vitro, the mutants displayed no phenotype in vivo, suggesting that permeation of nitrosative stress to the level of cytotoxic DNA damage is restricted.