Identification and in silico functional prediction of lineage-specific SNPs distributed in DosR-related proteins and resuscitation-promoting factor proteins of Mycobacterium tuberculosis.

One-third of the world population is infected by Mycobacterium tuberculosis, which may persist in the latent or dormant state. Bacteria can shift to dormancy when encountering harsh conditions such as low oxygen, nutrient starvation, high acidity and host immune defenses. Genes related to the dormancy survival regulator (DosR) regulon are responsible for the inhibition of aerobic respiration and replication, which is required to enter dormancy. Conversely, resuscitation-promoting factor (rpf) proteins participate in reactivation from dormancy and the development of active tuberculosis (TB). Many DosR regulon and rpf proteins are immunodominant T cell antigens that are highly expressed in latent TB infection. They could serve as TB vaccine candidates and be used for diagnostic development. We explored the genetic polymorphisms of 50 DosR-related genes and 5 rpf genes among 1,170 previously sequenced clinical M. tuberculosis genomes. Forty-three lineage- or sublineage-specific nonsynonymous single nucleotide polymorphisms (nsSNPs) were identified. Ten nsSNPs were specific to all Mtb isolates belonging to lineage 1 (L1). Two common sublineages, the Beijing family (L2.2) and EAI2 (L1.2.1), differed at as many as 26 lineage- or sublineage-specific SNPs. DosR regulon genes related to membrane proteins and the rpf family possessed mean dN/dS ratios greater than one, suggesting that they are under positive selection. Although the T cell epitope regions of DosR-related and rpf antigens were quite conserved, we found that the epitopes in L1 had higher rates of genetic polymorphisms than the other lineages. Some mutations in immunogenic epitopes of the antigens were specific to particular M. tuberculosis lineages. Therefore, the genetic diversity of the DosR regulon and rpf proteins might impact the adaptation of M. tuberculosis to the dormant state and the immunogenicity of latency antigens, which warrants further investigation.

Risk factors associated with large clusters of tuberculosis patients determined by whole-genome sequencing in a high-tuberculosis-burden country.

Whole-genome sequencing (WGS) analysis has great discriminative power for detecting similar molecular fingerprints of suspected tuberculosis (TB) clusters. The proportion of TB cases within clusters and the associated risk factors are important epidemiological parameters guiding appropriate outbreak control strategies in endemic settings. We conducted a hospital-based TB case-cohort study between 2003 and 2011 in the northernmost province of Thailand. We identified TB clusters by Mycobacterium tuberculosis WGS and analysed the risks of TB clustering and the characteristics of large clusters compared with small clusters. Among 1146 TB isolates, we identified 77 clusters with 251 isolates defined by a 5-single-nucleotide variant (SNV) cutoff and 112 clusters with 431 isolates defined by a 12-SNV cutoff. Twelve large clusters with 6 isolates or more in each cluster were identified by a 12-SNV cutoff. Sublineage 2.2.1 (both Ancestral and Modern) strains and imprisonment were independently associated with large clusters. Furthermore, although large clusters of Lineage 2.2.1/Ancestral strains included a high number of prisoners, Lineage 2.2.1/Modern strain clusters were only associated with treatment failures and drug resistance. Heterogeneity among lineage strains was observed with respect to large-cluster characteristics. Patients with an increased TB-transmission tendency should be priority targets for contact investigations and outbreak interventions to prevent ongoing transmission.

Homoplastic single nucleotide polymorphisms contributed to phenotypic diversity in Mycobacterium tuberculosis.

Homoplastic mutations are mutations independently occurring in different clades of an organism. The homoplastic changes may be a result of convergence evolution due to selective pressures. Reports on the analysis of homoplastic mutations in Mycobacterium tuberculosis have been limited. Here we characterized the distribution of homoplastic single nucleotide polymorphisms (SNPs) among genomes of 1,170 clinical M. tuberculosis isolates. They were present in all functional categories of genes, with pe/ppe gene family having the highest ratio of homoplastic SNPs compared to the total SNPs identified in the same functional category. Among the pe/ppe genes, the homoplastic SNPs were common in a relatively small number of homologous genes, including ppe18, the protein of which is a component of a promising candidate vaccine, M72/AS01E. The homoplastic SNPs in ppe18 were particularly common among M. tuberculosis Lineage 1 isolates, suggesting the need for caution in extrapolating the results of the vaccine trial to the population where L1 is endemic in Asia. As expected, homoplastic SNPs strongly associated with drug resistance. Most of these mutations are already well known. However, a number of novel mutations associated with streptomycin resistance were identified, which warrants further investigation. A SNP in the intergenic region upstream of Rv0079 (DATIN) was experimentally shown to increase transcriptional activity of the downstream gene, suggesting that intergenic homoplastic SNPs should have effects on the physiology of the bacterial cells. Our study highlights the potential of homoplastic mutations to produce phenotypic changes. Under selective pressure and during interaction with the host, homoplastic mutations may confer advantages to M. tuberculosis and deserve further characterization.

A novel Ancestral Beijing sublineage of Mycobacterium tuberculosis suggests the transition site to Modern Beijing sublineages.

Global Mycobacterium tuberculosis population comprises 7 major lineages. The Beijing strains, particularly the ones classified as Modern groups, have been found worldwide, frequently associated with drug resistance, younger ages, outbreaks and appear to be expanding. Here, we report analysis of whole genome sequences of 1170 M. tuberculosis isolates together with their patient profiles. Our samples belonged to Lineage 1-4 (L1-L4) with those of L1 and L2 being equally dominant. Phylogenetic analysis revealed several new or rare sublineages. Differential associations between sublineages of M. tuberculosis and patient profiles, including ages, ethnicity, HIV (human immunodeficiency virus) infection and drug resistance were demonstrated. The Ancestral Beijing strains and some sublineages of L4 were associated with ethnic minorities while L1 was more common in Thais. L2.2.1.Ancestral 4 surprisingly had a mutation that is typical of the Modern Beijing sublineages and was common in Akha and Lahu tribes who have migrated from Southern China in the last century. This may indicate that the evolutionary transition from the Ancestral to Modern Beijing sublineages might be gradual and occur in Southern China, where the presence of multiple ethnic groups might have allowed for the circulations of various co-evolving sublineages which ultimately lead to the emergence of the Modern Beijing strains.

Evidence for Host-Bacterial Co-evolution via Genome Sequence Analysis of 480 Thai Mycobacterium tuberculosis Lineage 1 Isolates.

Tuberculosis presents a global health challenge. Mycobacterium tuberculosis is divided into several lineages, each with a different geographical distribution. M. tuberculosis lineage 1 (L1) is common in the high-burden areas in East Africa and Southeast Asia. Although the founder effect contributes significantly to the phylogeographic profile, co-evolution between the host and M. tuberculosis may also play a role. Here, we reported the genomic analysis of 480 L1 isolates from patients in northern Thailand. The studied bacterial population was genetically diverse, allowing the identification of a total of 18 sublineages distributed into three major clades. The majority of isolates belonged to L1.1 followed by L1.2.1 and L1.2.2. Comparison of the single nucleotide variant (SNV) phylogenetic tree and the clades defined by spoligotyping revealed some monophyletic clades representing EAI2_MNL, EAI2_NTM and EAI6_BGD1 spoligotypes. Our work demonstrates that ambiguity in spoligotype assignment could be partially resolved if the entire DR region is investigated. Using the information to map L1 diversity across Southeast Asia highlighted differences in the dominant strain-types in each individual country, despite extensive interactions between populations over time. This finding supported the hypothesis that there is co-evolution between the bacteria and the host, and have implications for tuberculosis disease control.

Mutations in rrs, rpsL and gidB in streptomycin-resistant Mycobacterium tuberculosis isolates from Thailand.

The objectives of this study were to characterise mutations in rrs, rpsL and gidB genes in Mycobacterium tuberculosis isolates from Thailand and to examine possible associations between mutations and strain genotypes. In total, 110 streptomycin (STR)-resistant M. tuberculosis isolates and 51 STR-susceptible isolates obtained from a sample collection in Thailand during 1999-2011 were sequenced for mutation analysis in rrs, rpsL and gidB. Genotypes of the isolates were identified using spoligotyping and large sequence polymorphisms. Mutations at codons 43 and 88 in rpsL represented 63.6% of the STR-resistant isolates and were mostly associated with Beijing strains. Mutations in rrs existed in 17.3% of the STR-resistant isolates; only 8.2% harboured resistance-associated mutations. Twenty-five different mutations were found in gidB, twelve of which are new. Eight gidB mutations were likely to contribute to STR resistance in ca. 14% of the resistant isolates; about one-half of the isolates also had a mutation in rrs or rpsL. Nearly all of the double mutants belonged to Beijing strains, whereas isolates carrying only STR-associated gidB mutation were non-Beijing strains. Three different alleles in gidB were also found, each specific to Beijing, East-African Indian and Euro-American lineages, respectively. Most of the STR-resistant isolates (80.9%) carried putative resistance-associated mutations in the analysed genes. Beijing strains were related not only to single resistance-associated mutations in rpsL or rrs but usually harboured a second mutation in gidB. Strains harbouring resistance-associated gidB mutations without rrs or rpsL mutations were more associated with non-Beijing isolates. Certain gidB mutations were also potential lineage markers.

Expressed sequence tags reveal genetic diversity and putative virulence factors of the pathogenic oomycete Pythium insidiosum.

Oomycetes are unique eukaryotic microorganisms that share a mycelial morphology with fungi. Many oomycetes are pathogenic to plants, and a more limited number are pathogenic to animals. Pythium insidiosum is the only oomycete that is capable of infecting both humans and animals, and causes a life-threatening infectious disease, called "pythiosis". In the majority of pythiosis patients life-long handicaps result from the inevitable radical excision of infected organs, and many die from advanced infection. Better understanding P. insidiosum pathogenesis at molecular levels could lead to new forms of treatment. Genetic and genomic information is lacking for P. insidiosum, so we have undertaken an expressed sequence tag (EST) study, and report on the first dataset of 486 ESTs, assembled into 217 unigenes. Of these, 144 had significant sequence similarity with known genes, including 47 with ribosomal protein homology. Potential virulence factors included genes involved in antioxidation, thermal adaptation, immunomodulation, and iron and sterol binding. Effectors resembling pathogenicity factors of plant-pathogenic oomycetes were also discovered, such as, a CBEL-like protein (possible involvement in host cell adhesion and hemagglutination), a putative RXLR effector (possibly involved in host cell modulation) and elicitin-like (ELL) proteins. Phylogenetic analysis mapped P. insidiosum ELLs to several novel clades of oomycete elicitins (ELIs), and homology modeling predicted that P. insidiosum ELLs should bind sterols. Most of the P. insidiosum ESTs showed homology to sequences in the genome or EST databases of other oomycetes, but one putative gene, with unknown function, was found to be unique to P. insidiosum. The EST dataset reported here represents the first steps in identifying genes of P. insidiosum and beginning transcriptome analysis. This genetic information will facilitate understanding of pathogenic mechanisms of this devastating pathogen.

Intra-host diversities of the receptor-binding domain of stork faeces-derived avian H5N1 viruses and its significance as predicted by molecular dynamic simulation.

Virus evolution facilitates the emergence of viruses with unpredictable impacts on human health. This study investigated intra-host variations of the receptor-binding domain (RBD) of the haemagglutinin (HA) gene of the avian H5N1 viruses obtained from the 2004 and 2005 epidemics. The results showed that the mutation frequency of the RBD ranged from 0.3 to 0.6 %. The mutations generated one consensus and several minor populations. The consensus population of the 2004 epidemic was transmitted to the 2005 outbreak with increased frequency (39 and 45 %, respectively). Molecular dynamics simulation was applied to predict the significance of the variants. The results revealed that the consensus sequence (E218K/V248I) interacted unstably with sialic acid (SA) with an α2,6 linkage (SAα2,6Gal). Although the mutated K140R/E218K/V248I and Y191C/E218K/V248I sequences decreased the HA binding capacity to α2,3-linked SA, they were shown to bind α2,6-linked SA with increased affinity. Moreover, the substitutions at aa 140 and 191 were positive-selection sites. These data suggest that the K140R and Y191C mutations may represent a step towards human adaptation of the avian H5N1 virus.

Pulsed field gel electrophoresis analysis of Vibrio cholerae isolates in southern Thailand.

Forty isolates of V. cholorae O1, O139 and non-O1/non-O139 collected from outbreaks in Songkhla and Phuket Provinces of southern Thailand during 1999-2001 and sporadic cases from different regions of Thailand during 1993-2002 were characterized using pulsed field gel electrophoresis (PFGE). Digestion of chromosomal DNA of the V cholerae isolates with restriction endonuclease NotI, followed by PFGE, generated 10 distinct restriction endonuclease analysis patterns consisting of 8 to 13 bands, ranging in size from 78 to 394 kb. PFGE patterns of O1 Inaba strains from the outbreak in Songkhla were identical (P1) except one isolate (P3). The O1 Inaba outbreak strains from Phuket in the same period belonged to P2 pattern, whereas the O1 Ogawa strain from the outbreak in Phuket isolated in 1999 was of P7 pattern. These patterns of O1 Inaba and Ogawa strains were slightly different suggesting that the isolates were epidemiologically related and therefore the outbreaks were likely due to the same V cholerae clone. Isolates of V cholerae O1 Inaba from sporadic cases in the neighboring area (e.g., Pattani Province) in a similar period of time of the outbreak in Songkhla Province had very similar patterns, with only one single band different from those of the outbreak isolates. This indicates that the Inaba strains isolated from Songkhla Province during the 2001 cholera outbreak belonging to P1 pattern had not spread to other regions in 2001 and 2002. On the otherhand, the sporadic isolates collected from other regions of Thailand were quite distinct from the outbreak isolates in Songkhla Province, especially those from Chaiyaphum and Chaing Mai Provinces, which belonged to P5 and P6 pattern, respectively. Isolates of V cholerae O139 and non-O1/non-O139 gave different patterns from that of V. cholerae O1. This study shows that the PFGE technique is markedly advantageous in distinguishing strains of V cholerae isolates leading to insightful detailed charateristics of these isolates in Thailand.

The 74-kilodalton immunodominant antigen of the pathogenic oomycete Pythium insidiosum is a putative exo-1,3-beta-glucanase.

The oomycetous, fungus-like, aquatic organism Pythium insidiosum is the causative agent of pythiosis, a life-threatening infectious disease of humans and animals living in tropical and subtropical areas of the world. Common sites of infection are the arteries, eyes, cutaneous/subcutaneous tissues, and gastrointestinal tract. Diagnosis of pythiosis is time-consuming and difficult. Radical excision of the infected organs is the main treatment for pythiosis because conventional antifungal drugs are ineffective. An immunotherapeutic vaccine prepared from P. insidiosum crude extract showed limited efficacy in the treatment of pythiosis patients. Many pythiosis patients suffer lifelong disabilities or die from an advanced infection. Recently, we identified a 74-kDa major immunodominant antigen of P. insidiosum which could be a target for development of a more effective serodiagnostic test and vaccines. Mass spectrometric analysis identified two peptides of the 74-kDa antigen (s74-1 and s74-2) which perfectly matched a putative exo-1,3-ss-glucanase (EXO1) of Phytophthora infestans. Using degenerate primers derived from these peptides, a 1.1-kb product was produced by PCR, and its sequence was found to be homologous to that of the P. infestans exo-1,3-ss-glucanase gene, EXO1. Enzyme-linked immunosorbent assays targeting the s74-1 and s74-2 synthetic peptides demonstrated that the 74-kDa antigen was highly immunoreactive with pythiosis sera but not with control sera. Phylogenetic analysis using part of the 74-kDa protein-coding sequence divided 22 Thai isolates of P. insidiosum into two clades. Further characterization of the putative P. insidiosum glucanase could lead to new diagnostic tests and to antimicrobial agents and vaccines for the prevention and management of the serious and life-threatening disease of pythiosis.

Variable-number tandem repeats typing of Mycobacterium tuberculosis isolates with low copy numbers of IS6110 in Thailand.

Spoligotyping and variable-number tandem repeats (VNTR) typing have been increasingly used for differentiating Mycobacterium tuberculosis strains with low copy numbers of IS6110. However, there are few studies comparing their potential to type the strains originating from South and Southeast Asia where many of the isolates have only a few copies, or even single copy, of IS6110. Here, we evaluated the genotyping of 187M. tuberculosis isolates harboring 1-6 copies of IS6110, available from a population-based study in Chiangrai, northern Thailand during 1998-2000, using spoligotyping and VNTR typing. The low-copy-number isolates constituted about 34% of all M. tuberculosis isolated in the province. Discriminating capacities and cluster identification by the two methods were compared with each other and to those obtained by the standard IS6110-restriction fragment length polymorphism (RFLP) method. We found that VNTR typing based on the studied 10-loci set generated more distinct patterns (151 patterns) than spoligotyping (54 patterns) and IS6110-RFLP (65 patterns). Most of the RFLP- or spoligotyping-defined clusters were subdivided by VNTR typing. Combining IS6110-RFLP with VNTR typing produced 164 distinct patterns and 21.9% of clustered isolates whereas the combination of IS6110-RFLP and spoligotyping gave 103 different patterns and 59.4% of clustered isolates. Our results confirm the utility of VNTR typing as the secondary method of choice for investigating the epidemiology of M. tuberculosis with low copy numbers of IS6110.

Evolution of some variable-number tandem repeat loci among a group of Beijing strains of Mycobacterium tuberculosis.

The patterns of variable-number tandem repeats (VNTR) genotypes of a clonal group of Mycobacterium tuberculosis Beijing isolates with very similar IS6110-restriction fragment length polymorphism (RFLP) patterns were studied. Differences between VNTR were mostly by a single repeat unit. However, a multiple-unit change also occurred. This suggests that a mechanism other than the slipped-strand mispairing might be responsible for the instability of VNTR in M. tuberculosis as well. This finding is useful for inferring phylogenetics of M. tuberculosis based on the VNTR genotypes.