6 1 Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, 3010, 7 Australia. 8 2 Labortoratory for Antimicrobial Pharmacodynamics, School of Pharmacy and Pharmaceutical Sciences, 9 University of Buffalo, Buffalo, NY, USA. 10 3 Department of Microbiology, Monash University, Clayton, Victoria, 3800, Australia. 11 4 Austin Centre for Infection Research, Infectious Diseases Department, Austin Health, Heidelberg, Victoria, 12 3084, Australia. 13 5 Microbiology Department, Austin Health, Heidelberg, Victoria, 3084, Australia. 14 6 Victorian Bioinformatics Consortium, Monash University, Clayton, Victoria, 3800, Australia. 15

ABSTRACT Vancomycin-intermediate Staphylococcus aureus (VISA) strains often arise by mutations in the essential two-component regulator walKR ; however their impact on walKR function has not been definitively established. Here, we investigated 10 MRSA strains recovered serially after exposure of vancomycin-susceptible S. aureus (VSSA) JKD6009 to simulated human vancomycin dosing regimens (500 mg to 4,000 mg every 12 h) using a 10-day hollow fiber infection model. After continued exposure to the vancomycin regimens, two isolates displayed reduced susceptibility to both vancomycin and daptomycin, developing independent IS 256 insertions in the walKR 5′ untranslated region (5′ UTR). Quantitative reverse transcription-PCR (RT-PCR) revealed a 50% reduction in walKR gene expression in the IS 256 mutants compared to the VSSA parent. Green fluorescent protein (GFP) reporter analysis, promoter mapping, and site-directed mutagenesis confirmed these findings and showed that the IS 256 insertions had replaced two SigA-like walKR promoters with weaker, hybrid promoters. Removal of IS 256 reverted the phenotype to VSSA, showing that reduced expression of WalKR did induce the VISA phenotype. Analysis of selected WalKR-regulated autolysins revealed upregulation of ssaA but no change in expression of sak and sceD in both IS 256 mutants. Whole-genome sequencing of the two mutants revealed an additional IS 256 insertion within agrC for one mutant, and we confirmed that this mutation abolished agr function. These data provide the first substantial analysis of walKR promoter function and show that prolonged vancomycin exposure can result in VISA through an IS 256 -mediated reduction in walKR expression; however, the mechanisms by which this occurs remain to be determined.

Mycobacterium tuberculosis complex (MTBC) genomes contain 2 large gene families termed pe and ppe. The function of pe/ppe proteins remains enigmatic but studies suggest that they are secreted or cell surface associated and are involved in bacterial virulence. Previous studies have also shown that some pe/ppe genes are polymorphic, a finding that suggests involvement in antigenic variation. Using comparative sequence analysis of 18 publicly available MTBC whole genome sequences, we have performed alignments of 33 pe (excluding pe_pgrs) and 66 ppe genes in order to detect the frequency and nature of genetic variation. This work has been supplemented by whole gene sequencing of 14 pe/ppe (including 5 pe_pgrs) genes in a cohort of 40 diverse and well defined clinical isolates covering all the main lineages of the M. tuberculosis phylogenetic tree. We show that nsSNP's in pe (excluding pgrs) and ppe genes are 3.0 and 3.3 times higher than in non-pe/ppe genes respectively and that numerous other mutation types are also present at a high frequency. It has previously been shown that non-pe/ppe M. tuberculosis genes display a remarkably low level of purifying selection. Here, we also show that compared to these genes those of the pe/ppe families show a further reduction of selection pressure that suggests neutral evolution. This is inconsistent with the positive selection pressure of “classical” antigenic variation. Finally, by analyzing such a large number of genes we were able to detect large differences in mutation type and frequency between both individual genes and gene sub-families. The high variation rates and absence of selective constraints provides valuable insights into potential pe/ppe function. Since pe/ppe proteins are highly antigenic and have been studied as potential vaccine components these results should also prove informative for aspects of M. tuberculosis vaccine design.

Hanekom et al. ([1][1]) highlighted that host-pathogen compatibility determines the Beijing strain population structure in different host populations. Hanekom's study was based on 321 Mycobacterium tuberculosis strains, classified as Beijing/W by spoligotyping ([3][2]). These strains were genotyped

The Beijing strain is one of the most successful genotypes of Mycobacterium tuberculosis worldwide and appears to be highly homogenous according to existing genotyping methods. To type Beijing strains reliably we developed a robust typing scheme using single nucleotide polymorphisms (SNPs) and regions of difference (RDs) derived from whole-genome sequencing data of eight Beijing strains. SNP/RD typing of 259 M. tuberculosis isolates originating from 45 countries worldwide discriminated 27 clonal complexes within the Beijing genotype family. A total of 16 Beijing clonal complexes contained more than one isolate of known origin, of which two clonal complexes were strongly associated with South African origin. The remaining 14 clonal complexes encompassed isolates from different countries. Even highly resolved clonal complexes comprised isolates from distinct geographical sites. Our results suggest that Beijing strains spread globally on multiple occasions and that the tuberculosis epidemic caused by the Beijing genotype is at least partially driven by modern migration patterns. The SNPs and RDs presented in this study will facilitate future molecular epidemiological and phylogenetic studies on Beijing strains.

Precise annotation of genes or open reading frames is still a difficult task that results in divergence even for data generated from the same genomic sequence. This has an impact in further proteomic studies, and also compromises the characterization of clinical isolates with many specific genetic variations that may not be represented in the selected database. We recently developed software called multistrain mass spectrometry prokaryotic database builder (MSMSpdbb) that can merge protein databases from several sources and be applied on any prokaryotic organism, in a proteomic-friendly approach. We generated a database for the Mycobacterium tuberculosis complex (using three strains of Mycobacterium bovis and five of M. tuberculosis), and analyzed data collected from two laboratory strains and two clinical isolates of M. tuberculosis. We identified 2561 proteins, of which 24 were present in M. tuberculosis H37Rv samples, but not annotated in the M. tuberculosis H37Rv genome. We were also able to identify 280 nonsynonymous single amino acid polymorphisms and confirm 367 translational start sites. As a proof of concept we applied the database to whole-genome DNA sequencing data of one of the clinical isolates, which allowed the validation of 116 predicted single amino acid polymorphisms and the annotation of 131 N-terminal start sites. Moreover we identified regions not present in the original M. tuberculosis H37Rv sequence, indicating strain divergence or errors in the reference sequence. In conclusion, we demonstrated the potential of using a merged database to better characterize laboratory or clinical bacterial strains.

RATIONALE:Central dogma suggests that rifampicin resistance in Mycobacterium tuberculosis develops solely through rpoB gene mutations.OBJECTIVE:To determine whether rifampicin induces efflux pumps activation in rifampicin resistant M. tuberculosis strains thereby defining rifampicin resistance levels and reducing ofloxacin susceptibility.METHODS:Rifampicin and/or ofloxacin minimum inhibitory concentrations (MICs) were determined in rifampicin resistant strains by culture in BACTEC 12B medium. Verapamil and reserpine were included to determine their effect on rifampicin and ofloxacin susceptibility. RT-qPCR was applied to assess expression of efflux pump/transporter genes after rifampicin exposure. To determine whether verapamil could restore susceptibility to first-line drugs, BALB/c mice were infected with a MDR-TB strain and treated with first-line drugs with/without verapamil.MEASUREMENTS AND MAIN FINDINGS:Rifampicin MICs varied independently of rpoB mutation and genetic background. Addition reserpine and verapamil significantly restored rifampicin susceptibility (p = 0.0000). RT-qPCR demonstrated that rifampicin induced differential expression of efflux/transporter genes in MDR-TB isolates. Incubation of rifampicin mono-resistant strains in rifampicin (2 μg/ml) for 7 days induced ofloxacin resistance (MIC > 2 μg/ml) in strains with an rpoB531 mutation. Ofloxacin susceptibility was restored by exposure to efflux pump inhibitors. Studies in BALB/c mice showed that verapamil in combination with first-line drugs significantly reduced pulmonary CFUs after 1 and 2 months treatment (p < 0.05).CONCLUSION:Exposure of rifampicin resistant M. tuberculosis strains to rifampicin can potentially compromise the efficacy of the second-line treatment regimens containing ofloxacin, thereby emphasising the need for rapid diagnostics to guide treatment. Efflux pump inhibitors have the potential to improve the efficacy of anti-tuberculosis drug treatment.

Antimicrobial resistance in Staphylococcus aureus is a major public health threat, compounded by emergence of strains with resistance to vancomycin and daptomycin, both last line antimicrobials. Here we have performed high throughput DNA sequencing and comparative genomics for five clinical pairs of vancomycin-susceptible (VSSA) and vancomycin-intermediate ST239 S. aureus (VISA); each pair isolated before and after vancomycin treatment failure. These comparisons revealed a frequent pattern of mutation among the VISA strains within the essential walKR two-component regulatory locus involved in control of cell wall metabolism. We then conducted bi-directional allelic exchange experiments in our clinical VSSA and VISA strains and showed that single nucleotide substitutions within either walK or walR lead to co-resistance to vancomycin and daptomycin, and caused the typical cell wall thickening observed in resistant clinical isolates. Ion Torrent genome sequencing confirmed no additional regulatory mutations had been introduced into either the walR or walK VISA mutants during the allelic exchange process. However, two potential compensatory mutations were detected within putative transport genes for the walK mutant. The minimal genetic changes in either walK or walR also attenuated virulence, reduced biofilm formation, and led to consistent transcriptional changes that suggest an important role for this regulator in control of central metabolism. This study highlights the dramatic impacts of single mutations that arise during persistent S. aureus infections and demonstrates the role played by walKR to increase drug resistance, control metabolism and alter the virulence potential of this pathogen.

Although the genome of the Mycobacterium tuberculosis H37Rv laboratory strain has been available for over 10 years, it is only recently that genomic information from clinical isolates has been used to generate the hypothesis of virulence differences between different strains. In addition, the relationship between strains displaying differing virulence in an epidemiological setting and their behavior in animal models has received little attention. The potential causes for variation in virulence between strains, as determined by differential protein expression, have similarly been a neglected area of investigation. In this study, we used a label-free quantitative proteomics approach to estimate differences in protein abundance between two closely related Beijing genotypes that have been shown to be hyper- and hypovirulent on the basis of both epidemiological and mouse model studies. We were able to identify a total of 1668 proteins from both samples, and protein abundance calculations revealed that 48 proteins were over-represented in the hypovirulent isolate, whereas 53 were over-represented in the hypervirulent. Functional classification of these results shows that molecules of cell wall organization and DNA transcription regulatory proteins may have a critical influence in defining the level of virulence. The reduction in the presence of ESAT-6, other Esx-like proteins, and FbpD (MPT51) in the hypervirulent strain indicates that changes in the repertoire of highly immunogenic proteins can be a defensive process undertaken by the virulent cell. In addition, most of the previously well characterized gene targets related to virulence were found to be similarly expressed in our model. Our data support the use of proteomics as a complementary tool for genomic comparisons to understand the biology of M. tuberculosis virulence.

Separating rat pups from their mothers during the early stages of life is an animal model commonly used to study the development of psychiatric disorders such as anxiety and depression. The present study investigated how soon after the termination of the maternal separation period behavioural and neuroendocrine abnormalities relevant to above-mentioned illnesses would manifest. Sprague Dawley rat pups were subjected to maternal separation (3 h per day from postnatal day 2 through 14) and their behaviour and HPA axis activity determined 7 d later. We also measured nerve growth factor levels in their hippocampi and assessed the DNA methylation status of the promoter region of exon 17 of the glucocorticoid receptor in this brain region. As early as 7 d after the termination of the adverse event, a change in behaviour was observed that was associated with increased plasma corticosterone release and elevated nerve growth factor levels in the hippocampus. No alteration in the methylation status of the exon 17 glucocorticoid receptor promoter region was observed. Our data indicate that early life adversity may lead to the rapid development of abnormal behaviours and HPA axis dysregulation though no epigenetic changes to the exon 17 glucocorticoid receptor promoter region occurred. We further propose that the observed increased neurotrophin levels reflect compensatory mechanisms that attempt to combat the long-term deleterious effects of maternal separation.

IS6110 is a transposable element found in Mycobacterium tuberculosis complex members. Regions of preferential IS6110 integration occur within the M. tuberculosis genome but the element has not previously been shown to exhibit any sequence-specific integration preferences. Here we provide evidence for multiple independent IS6110 insertions into identical, or near-identical, positions within the highly homologous 5′ region of 3 PPE genes.

Separating rat pups from their mothers during the early stages of life is an animal model commonly used to study the development of psychiatric disorders such as anxiety and depression. The present study investigated how soon after the termination of the maternal separation period behavioural and neuroendocrine abnormalities relevant to above-mentioned illnesses would manifest. Sprague Dawley rat pups were subjected to maternal separation (3 h per day from postnatal day 2 through 14) and their behaviour and HPA axis activity determined 7 d later. We also measured nerve growth factor levels in their hippocampi and assessed the DNA methylation status of the promoter region of exon 1 7 of the glucocorticoid receptor in this brain region. As early as 7 d after the termination of the adverse event, a change in behaviour was observed that was associated with increased plasma corticosterone release and elevated nerve growth factor levels in the hippocampus. No alteration in the methylation status of the exon 1 7 glucocorticoid receptor promoter region was observed. Our data indicate that early life adversity may lead to the rapid development of abnormal behaviours and HPA axis dysregulation though no epigenetic changes to the exon 1 7 glucocorticoid receptor promoter region occurred. We further propose that the observed increased neurotrophin levels reflect compensatory mechanisms that attempt to combat the long-term deleterious effects of maternal separation.

BACKGROUND:PPE38 (Rv2352c) is a member of the large PPE gene family of Mycobacterium tuberculosis and related mycobacteria. The function of PPE proteins is unknown but evidence suggests that many are cell-surface associated and recognised by the host immune system. Previous studies targeting other PPE gene members suggest that some display high levels of polymorphism and it is thought that this might represent a means of providing antigenic variation. We have analysed the genetic variability of the PPE38 genomic region on a cohort of M. tuberculosis clinical isolates representing all of the major phylogenetic lineages, along with the ancestral M. tuberculosis complex (MTBC) member M. canettii, and supplemented this with analysis of publicly available whole genome sequences representing additional M. tuberculosis clinical isolates, other MTBC members and non tuberculous mycobacteria (NTM). Where possible we have extended this analysis to include the adjacent plcABC and PPE39/40 genomic regions.RESULTS:We show that the ancestral MTBC PPE38 region comprises 2 homologous PPE genes (PPE38 and PPE71), separated by 2 esat-6 (esx)-like genes and that this structure derives from an esx/esx/PPE duplication in the common ancestor of M. tuberculosis and M. marinum. We also demonstrate that this region of the genome is hypervariable due to frequent IS6110 integration, IS6110-associated recombination, and homologous recombination and gene conversion events between PPE38 and PPE71. These mutations result in combinations of gene deletion, gene truncation and gene disruption in the majority of clinical isolates. These mutations were generally found to be IS6110 strain lineage-specific, although examples of additional within-lineage and even within-cluster mutations were observed. Furthermore, we provide evidence that the published M. tuberculosis H37Rv whole genome sequence is inaccurate regarding this region.CONCLUSION:Our results show that this antigen-encoding region of the M. tuberculosis genome is hypervariable. The observation that numerous different mutations have become fixed within specific lineages demonstrates that this genomic region is undergoing rapid molecular evolution and that further lineage-specific evolutionary expansion and diversification has occurred subsequent to the lineage-defining mutational events. We predict that functional loss of these genes could aid immune evasion. Finally, we also show that the PPE38 region of the published M. tuberculosis H37Rv whole genome sequence is not representative of the ATCC H37Rv reference strain.

Since the discovery of the tubercle bacillus by Robert Koch in 1882 ([110][1]), a greater understanding of the dynamics and survival mechanisms of this pathogen has led to more questions than answers. Despite stringent control strategies and many advances in our knowledge of the epidemiology of

ABSTRACT IS 6110 restriction fragment length polymorphism (RFLP) genotyping is the most widely used genotyping method to study the epidemiology of Mycobacterium tuberculosis . However, due to the complexity of the IS 6110 RFLP genotyping technique, and the interpretation of RFLP data, mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) genotyping has been proposed as the new genotyping standard. This study aimed to determine the discriminatory power of different MIRU-VNTR locus combinations relative to IS 6110 RFLP genotyping, using a collection of Beijing genotype M. tuberculosis strains with a well-established phylogenetic history. Clustering, diversity index, clustering concordance, concordance among unique genotypes, and divergent and convergent evolution were calculated for seven combinations of 27 different MIRU-VNTR loci and compared to IS 6110 RFLP results. Our results confirmed previous findings that MIRU-VNTR genotyping can be used to estimate the extent of recent or ongoing transmission. However, molecular epidemiological linking of cases varied significantly depending on the genotyping method used. We conclude that IS 6110 RFLP and MIRU-VNTR loci evolve independently and at different rates, which leads to discordance between transmission chains predicted by the respective genotyping methods. Concordance between the two genotyping methods could be improved by the inclusion of genetic distance (GD) into the clustering formulae for some of the MIRU-VNTR loci combinations. In summary, our findings differ from previous reports, which may be explained by the fact that in settings of low tuberculosis incidence, the genetic distance between epidemiologically unrelated isolates was sufficient to define a strain using either marker, whereas in settings of high incidence, continuous evolution and persistence of strains revealed the weaknesses inherent to these markers.

The importance of core histones in the regulation of DNA function by chromatin is clear. However, little is known about the role of the linker histone. We investigated the role of H1 in Saccharomyces cerevisiae during extensive transcriptional reprogramming in stationary phase. Although the levels of linker histone Hho1p remained constant during growth to semiquiescence, there was a genome-wide increase in binding to chromatin. Hho1p was essential for compaction of chromatin in stationary phase, but not for general transcriptional repression. A clear, genome-wide anticorrelation was seen between the level of bound Hho1p and gene expression. Surprisingly, the rank order of gene activity was maintained even in the absence of Hho1p. Based on these findings, we suggest that linker histone Hho1p has a limited role in transcriptional regulation and that the dynamically exchanging linker histone may be evicted from chromatin by transcriptional activity.

Members of the Mycobacterium tuberculosis complex contain the transposable element IS6110 which, due to its high numerical and positional polymorphism, has become a widely used marker in epidemiological studies. Here, we review the evidence that IS6110 is not simply a passive or 'junk' DNA sequence, but that, through its transposable activity, it is able to generate genotypic variation that translates into strain-specific phenotypic variation. We also speculate on the role that this variation has played in the evolution of M. tuberculosis and conclude that the presence of a moderate IS6110 copy number within the genome may provide the pathogen with a selective advantage that has aided its virulence.

ABSTRACT This study aimed to reconstruct the evolutionary history of Beijing strains of Mycobacterium tuberculosis and to test the hypothesis that evolution has influenced the ability of the Beijing strains within the different Beijing sublineages to spread and cause disease. A PCR-based method was used to analyze the genome structure of 40 different loci in 325 Beijing isolates collected from new and retreatment tuberculosis patients from an urban setting and 270 Beijing isolates collected from high-risk tuberculosis patients from a rural setting in the Western Cape, South Africa. The resulting data were subjected to phylogenetic analysis using the neighbor joining algorithm. Phylogenetic reconstructions were highly congruent with the “gold standard” phylogenetic tree based on synonymous single-nucleotide polymorphisms, thereby allowing a prediction of the order in which the evolutionary events had occurred. A total of seven independently evolving Beijing sublineages were identified. Analysis of epidemiological data in relation to the Beijing sublineage suggested an association between recent evolutionary change and frequency of occurrence in an urban population (P < 0.001) as well as in the rural population (P < 0.001). This concept was further supported by an association between more recently evolved Beijing strains and an increased ability to transmit and to cause disease (odds ratio, 5.82; 95% confidence interval, 3.13 to 10.82 [P < 0.001]). An association between Beijing sublineage and demographic and clinical parameters and drug resistance could not be demonstrated. From these data, we suggest that the pathogenic characteristics of Beijing strains are not conserved but rather that strains within individual lineages have evolved unique pathogenic characteristics.

This study describes a comparative analysis of the Beijing mycobacterial interspersed repetitive unit types of Mycobacterium tuberculosis isolates from Cape Town, South Africa, and East Asia. The results show a significant association between the frequency of occurrence of strains from defined Beijing sublineages and the human population from whom they were cultured (P < 0.0001).