Nicolaas C. Gey van Pittius

Evolution and expansion of the Mycobacterium tuberculosis PE and PPE multigene families and their association with the duplication of the ESAT-6 (esx) gene cluster regions.

BackgroundThe PE and PPE multigene families of Mycobacterium tuberculosis comprise about 10% of the coding potential of the genome. The function of the proteins encoded by these large gene families remains unknown, although they have been proposed to be involved in antigenic variation and disease pathogenesis. Interestingly, some members of the PE and PPE families are associated with the ESAT-6 (esx) gene cluster regions, which are regions of immunopathogenic importance, and encode a system dedicated to the secretion of members of the potent T-cell antigen ESAT-6 family. This study investigates the duplication characteristics of the PE and PPE gene families and their association with the ESAT-6 gene clusters, using a combination of phylogenetic analyses, DNA hybridization, and comparative genomics, in order to gain insight into their evolutionary history and distribution in the genus Mycobacterium.ResultsThe results showed that the expansion of the PE and PPE gene families is linked to the duplications of the ESAT-6 gene clusters, and that members situated in and associated with the clusters represent the most ancestral copies of the two gene families. Furthermore, the emergence of the repeat protein PGRS and MPTR subfamilies is a recent evolutionary event, occurring at defined branching points in the evolution of the genus Mycobacterium. These gene subfamilies are thus present in multiple copies only in the members of the M. tuberculosis complex and close relatives. The study provides a complete analysis of all the PE and PPE genes found in the sequenced genomes of members of the genus Mycobacterium such as M. smegmatis, M. avium paratuberculosis, M. leprae, M. ulcerans, and M. tuberculosis.ConclusionThis work provides insight into the evolutionary history for the PE and PPE gene families of the mycobacteria, linking the expansion of these families to the duplications of the ESAT-6 (esx) gene cluster regions, and showing that they are composed of subgroups with distinct evolutionary (and possibly functional) differences.

Systematic Genetic Nomenclature for Type VII Secretion Systems

CITATION: Bitter, W., et al. 2009. Systematic genetic nomenclature for type VII secretion systems. PLoS Pathogens, 5(10): 1-6, doi: 10.1371/journal.ppat.1000507.

PPE and PE_PGRS proteins of Mycobacterium marinum are transported via the type VII secretion system ESX-5

ESX‐5 is one of the five type VII secretion systems found in mycobacteria. These secretion systems are also known as ESAT‐6‐like secretion systems. Here, we have determined the secretome of ESX‐5 by a proteomic approach in two different strains of Mycobacterium marinum. Comparison of the secretion profile of wild‐type strains and their ESX‐5 mutants showed that a number of PE_PGRS and PPE‐MPTR proteins are dependent on ESX‐5 for transport. The PE and PPE protein families are unique to mycobacteria, are highly expanded in several pathogenic species, such as Mycobacterium tuberculosis and M. marinum, and certain family members are cell surface antigens associated with virulence. Using a monoclonal antibody directed against the PGRS domain we showed that nearly all PE_PGRS proteins that are recognized by this antibody are missing in the supernatant of ESX‐5 mutants. In addition to PE_PGRS and PPE proteins, the ESX‐5 secretion system is responsible for the secretion of a ESAT‐6‐like proteins. Together, these data show that ESX‐5 is probably a major secretion pathway for mycobacteria and that this system is responsible for the secretion of recently evolved PE_PGRS and PPE proteins.

Novel Mycobacterium tuberculosis Complex Pathogen, M. mungi

Seven outbreaks involving increasing numbers of banded mongoose troops and high death rates have been documented. We identified a Mycobacterium tuberculosis complex pathogen, M. mungi sp. nov., as the causative agent among banded mongooses that live near humans in Chobe District, Botswana. Host spectrum and transmission dynamics remain unknown.

Emergence and Spread of Extensively and Totally Drug-Resistant Tuberculosis, South Africa

Factors driving the increase in drug-resistant tuberculosis (TB) in the Eastern Cape Province, South Africa, are not understood. A convenience sample of 309 drug-susceptible and 342 multidrug-resistant (MDR) TB isolates, collected July 2008–July 2009, were characterized by spoligotyping, DNA fingerprinting, insertion site mapping, and targeted DNA sequencing. Analysis of molecular-based data showed diverse genetic backgrounds among drug-sensitive and MDR TB sensu stricto isolates in contrast to restricted genetic backgrounds among pre–extensively drug-resistant (pre-XDR) TB and XDR TB isolates. Second-line drug resistance was significantly associated with the atypical Beijing genotype. DNA fingerprinting and sequencing demonstrated that the pre-XDR and XDR atypical Beijing isolates evolved from a common progenitor; 85% and 92%, respectively, were clustered, indicating transmission. Ninety-three percent of atypical XDR Beijing isolates had mutations that confer resistance to 10 anti-TB drugs, and some isolates also were resistant to para-aminosalicylic acid. These findings suggest the emergence of totally drug-resistant TB.

Rifampicin Reduces Susceptibility to Ofloxacin in Rifampicin-resistant Mycobacterium tuberculosis through Efflux

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.

Application of Sensitive and Specific Molecular Methods To Uncover Global Dissemination of the Major RDRio Sublineage of the Latin American-Mediterranean Mycobacterium tuberculosis Spoligotype Family

ABSTRACT The Latin American-Mediterranean (LAM) family of Mycobacterium tuberculosis is believed to be the cause of ∼15% of tuberculosis cases worldwide. Previously, we defined a prevalent sublineage of the LAM family in Brazil by a single characteristic genomic deletion designated RDRio. Using the Brazilian strains, we pinpoint an Ag85C103 single nucleotide polymorphism (SNP) (screened by restriction fragment length polymorphism [RFLP] analysis) that correctly identified all LAM family strains. Importantly, all RDRio strains concomitantly possessed the RD174 deletion. These genetic signatures, along with a newly developed multiplex PCR for rapid differentiation between “wild-type” and RDRio strains, were then used to analyze an international collection of M. tuberculosis strains. RDRioM. tuberculosis was identified from four continents involving 11 countries. Phylogenetic analysis of the IS6110-RFLP patterns from representative RDRio and LAM strains from Brazil, along with all representative clusters from a South African database, confirmed their genetic relatedness and transcontinental transmission. The Ag85C103 SNP RFLP, as compared to results obtained using a PCR method targeting a LAM-restricted IS6110 element, correctly identified 99.8% of LAM spoligotype strains. Together, these tests were more accurate than spoligotyping at categorizing strains with indefinable spoligotypes and segregated true LAM strains from those with convergent spoligotypes. The fact that RDRio strains were identified worldwide highlights the importance of this LAM family sublineage and suggests that this strain is a global threat that should be specifically targeted by public health resources. Our provision of simple and robust molecular methods will assist the evaluation of the LAM family and the RDRio sublineage.

The non-clonality of drug resistance in Beijing-genotype isolates of Mycobacterium tuberculosis from the Western Cape of South Africa.

BackgroundThe Beijing genotype of M. tuberculosis is a virulent strain that is disseminating worldwide and has a strong association with drug resistance. In the Western Cape of South Africa, epidemiological studies have identified the R220 cluster of the Beijing genotype as a major contributor to a recent outbreak of drug-resistant tuberculosis. Although the outbreak is considered to be due to clonal transmission, the relationship among drug resistant isolates has not yet been established.ResultsTo better understand the evolution of drug resistance among these strains, 14 drug-resistant clinical isolates of the Beijing genotype were sequenced by whole-genome sequencing, including eight from R220 and six from a more ancestral Beijing cluster, R86, for comparison. While each cluster shares a distinct resistance mutation for isoniazid, mapping of other drug-resistance mutations onto a phylogenetic tree constructed from single nucleotide polymorphisms shows that resistance mutations to many drugs have arisen multiple times independently within each cluster of isolates. Thus, drug resistance among these isolates appears to be acquired, not clonally derived. This observation suggests that, although the Beijing genotype as a whole might have selective advantages enabling its rapid dissemination, the XDR isolates are relatively less fit and do not propagate well. Although it has been hypothesized that the increased frequency of drug resistance in some Beijing lineages might be caused by a mutator phenotype, no significant shift in synonymous substitution patterns is observed in the genomes.ConclusionWhile MDR-TB is spreading by transmission in the Western Cape, our data suggests that further drug resistance (i.e. XDR-TB) at this stage is acquired.

Evidence for a rapid rate of molecular evolution at the hypervariable and immunogenic Mycobacterium tuberculosis PPE38 gene region

BackgroundPPE38 (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.ResultsWe 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.ConclusionOur 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.

Frequent Homologous Recombination Events in Mycobacterium tuberculosis PE/PPE Multigene Families: Potential Role in Antigenic Variability

The PE and PPE (PE/PPE) multigene families of Mycobacterium tuberculosis are particularly GC-rich and share extensive homologous repetitive sequences. We hypothesized that they may undergo homologous recombination events, a mechanism rarely described in the natural evolution of mycobacteria. To test our hypothesis, we developed a specific oligonucleotide-based microarray targeting nearly all of the PE/PPE genes, aimed at detecting signals for homologous recombination. Such a microarray has never before been reported due to the multiplicity and highly repetitive and homologous nature of these sequences. Application of the microarray to a collection of M. tuberculosis clinical isolates (n = 33) representing prevalent spoligotype strain families in Tunisia allowed successful detection of six deleted genomic regions involving a total of two PE and seven PPE genes. Some of these deleted genes are known to be immunodominant or involved in virulence. The four precisely determined deletions were flanked by 400- to 500-bp stretches of nearly identical sequences lying mainly at the conserved N-terminal region of the PE/PPE genes. These highly homologous sequences thus serve as substrates to mediate both intergenic and intragenic homologous recombination events, indicating an important function in generating strain variation. Importantly, all recombination events yielded a new in-frame fusion chimeric gene. Hence, homologous recombination within and between PE/PPE genes likely increased their antigenic variability, which may have profound implications in pathogenicity and/or host adaptation. The finding of high prevalence (approximately 45% and approximately 58%) for at least two of the genomic deletions suggests that they likely confer advantageous biological attributes.