Sarah Talarico

Execution of macrophage apoptosis by PE_PGRS33 of Mycobacterium tuberculosis is mediated by toll-like receptor 2-dependent release of tumor necrosis factor-α

Combating tuberculosis requires a detailed understanding of how mycobacterial effectors modulate the host immune response. The role of the multigene PE family of proteins unique to mycobacteria in the pathogenesis of tuberculosis is still poorly understood, although certain PE_PGRS genes have been linked to virulence. Tumor necrosis factor-α (TNF-α) is essential for successfully combating tuberculosis. In this study we provide evidence that PE_PGRS33, a surface exposed protein, elicits TNF-α release from macrophages in a TLR2 (Toll-like receptor 2)-dependent manner. ASK1 (apoptosis signal-regulating kinase 1) is activated downstream of TLR2. ASK1 activates the MAPKs p38 and JNK. PE_PGRS33-induced signaling leads to enhanced expression of TNF-α and TNF receptor I (TNFRI) genes. Mycobacterium smegmatis expressing PE_ PGRS33 elicits the same effects as purified PE_PGRS33. TNF-α release occurs even when internalization of the bacteria is blocked by cytochalasin D, suggesting that interaction of PE_ PGRS33 with TLR2 is sufficient to trigger the effects described. Release of TNF-α plays the determining role in triggering apoptosis in macrophages challenged with PE_PGRS33. The death receptor-dependent signals are amplified through classical caspase 8-dependent mitochondrial release of cytochrome c, leading to the activation of caspases 9 and 3. An important aspect of our findings is that deletions within the PGRS domain (simulating those occurring in clinical strains) attenuate the TNF-α-inducing ability of PE_PGRS33. These results provide the first evidence that variations in the polymorphic repeats of the PGRS domain modulate the innate immune response.

Helicobacter pylori's unconventional role in health and disease.

The discovery of a bacterium, Helicobacter pylori, that is resident in the human stomach and causes chronic disease (peptic ulcer and gastric cancer) was radical on many levels. Whereas the mouth and the colon were both known to host a large number of microorganisms, collectively referred to as the microbiome, the stomach was thought to be a virtual Sahara desert for microbes because of its high acidity. We now know that H. pylori is one of many species of bacteria that live in the stomach, although H. pylori seems to dominate this community. H. pylori does not behave as a classical bacterial pathogen: disease is not solely mediated by production of toxins, although certain H. pylori genes, including those that encode exotoxins, increase the risk of disease development. Instead, disease seems to result from a complex interaction between the bacterium, the host, and the environment. Furthermore, H. pylori was the first bacterium observed to behave as a carcinogen. The innate and adaptive immune defenses of the host, combined with factors in the environment of the stomach, apparently drive a continuously high rate of genomic variation in H. pylori. Studies of this genetic diversity in strains isolated from various locations across the globe show that H. pylori has coevolved with humans throughout our history. This long association has given rise not only to disease, but also to possible protective effects, particularly with respect to diseases of the esophagus. Given this complex relationship with human health, eradication of H. pylori in nonsymptomatic individuals may not be the best course of action. The story of H. pylori teaches us to look more deeply at our resident microbiome and the complexity of its interactions, both in this complex population and within our own tissues, to gain a better understanding of health and disease.

Variation of the Mycobacterium tuberculosis PE_PGRS33 Gene among Clinical Isolates

ABSTRACT PE_PGRS33, one of about 60 PE_PGRS genes in the Mycobacterium tuberculosis genome, encodes a surface-expressed protein that may be involved in the antigenic variation of M. tuberculosis strains and evasion of the host immune system. While genetic differences between the PE_PGRS33 genes of H37Rv and CDC1551 have been noted, genetic variation in this gene among clinical isolates has not been evaluated. In order to gain a better understanding of the genetic basis for the role of PE_PGRS in antigenic variation and evasion of the host immune system, we investigated the genetic diversity of the PE_PGRS33 gene among 123 clinical M. tuberculosis isolates from a population-based study, using PCR and DNA sequencing. The 123 isolates belonged to principal genetic groups 1, 2, and 3 and had IS6110 copy numbers ranging from 1 to 22. Eighty-four (68.3%) of the 123 isolates were found to have at least one sequence variation in the PE_PGRS33 gene, relative to that of H37Rv. Twenty-five different sequence variations were observed and included three insertions (ranging from 9 to 87 bp), nine deletions (ranging from 1 to 273 bp), one insertion-and-deletion event, and 12 single-nucleotide polymorphisms (six synonymous and six nonsynonymous). Analysis of the relationships among the different PE_PGRS33 gene sequence variations suggests that polymorphisms in the gene are shifting along evolutionary lineages. The observed genetic diversity of the PE_PGRS33 gene supports its role in antigenic variation and can serve as a basis for future investigations of the function of the PE_PGRS33 gene among clinical isolates.

Mycobacterium tuberculosis PE_PGRS16 and PE_PGRS26 genetic polymorphism among clinical isolates

The Mycobacterium tuberculosis PE_PGRS multigene family is thought to be involved in antigenic variation, which can be generated by differential regulation of expression and a high frequency of genetic polymorphism. PE_PGRS16 and PE_PGRS26 are inversely regulated during persistent M. tuberculosis infection, suggesting that differential regulation of the expression of these two PE_PGRS genes may have a role in latency. To understand how genetic diversity, in addition to differential regulation, contributes to antigenic variability, we investigated the sequence variations in the PE_PGRS16 and PE_PGRS26 genes among 200 clinical M. tuberculosis strains, in comparison to the sequenced laboratory strain H37Rv, using PCR and DNA sequencing. Among the 200 strains, 102 (51%) and 100 (50%) had sequence variations within the PE_PGRS16 gene and the PE_PGRS26 gene, respectively. In-frame insertions and deletions, frameshifts, and SNPs were observed in both the PE_PGRS16 gene and the PE_PGRS26 gene. However, the frequency of frameshifts and in-frame deletions differed between the two PE_PGRS genes. Examining the profile of the PE_PGRS16, PE_PGRS26, and the previously investigated PE_PGRS33 amino acid sequences for each of the 200 strains, 72 different profiles were observed with frequencies ranging from 0.5% to 13%. In conclusion, a remarkable level of genetic diversity exists in the PE_PGRS16 and PE_PGRS26 genes of M. tuberculosis clinical strains. The significant sequence variations in the two PE_PGRS genes observed in this study could impact the function of these two PE_PGRS proteins and be associated with differences in the ability of the tubercle bacilli to remain persistent within the host.

Regulation of Helicobacter pylori adherence by gene conversion.

Genetic diversification of Helicobacter pylori adhesin genes may allow adaptation of adherence properties to facilitate persistence despite host defences. The sabA gene encodes an adhesin that binds sialyl‐Lewis antigens on inflamed gastric tissue. We found variability in the copy number and locus of the sabA gene and the closely related sabB and omp27 genes due to gene conversion among 51 North American paediatric H. pylori strains. We determined that sabB to sabA gene conversion is predominantly the result of intra‐genomic recombination and RecA, RecG and AddA influence the rate at which it occurs. Although all clinical strains had at least one sabA gene copy, sabA and sabB were lost due to gene conversion at similar rates in vitro, suggesting host selection to maintain the sabA gene. sabA gene duplication resulted in increased SabA protein production and increased adherence to sialyl‐Lewis antigens and mouse gastric tissue. In conclusion, gene conversion is a mechanism for H. pylori to regulate sabA expression level and adherence.

Pediatric Helicobacter pylori Isolates Display Distinct Gene Coding Capacities and Virulence Gene Marker Profiles

ABSTRACT Helicobacter pylori strains display remarkable genetic diversity, and the presence of strains bearing the toxigenic vacA s1 allele, a complete cag pathogenicity island (PAI), cagA alleles containing multiple EPIYA phosphorylation sites, and expressing the BabA adhesin correlates with development of gastroduodenal disease in adults. To better understand the genetic variability present among pediatric strains and its relationship to disease, we characterized H. pylori strains infecting 47 pediatric North American patients. Prevalence of mixed infection was assessed by random amplified polymorphic DNA analysis of multiple H. pylori clones from each patient. Microarray-based comparative genomic hybridization was used to examine the genomic content of the pediatric strains. The cagA and vacA alleles were further characterized by allele-specific PCR. A range of EPIYA motif configurations were observed for the cagA gene, which was present in strains from 22 patients (47%), but only 19 (41%) patients contained a complete cag PAI. Thirty patients (64%) were infected with a strain having the vacA s1 allele, and 28 patients (60%) had the babA gene. The presence of a functional cag PAI was correlated with ulcer disease (P = 0.0095). In spite of declining rates of H. pylori infection in North America, at least 11% of patients had mixed infection. Pediatric strains differ in their spectrum of strain-variable genes and percentage of absent genes in comparison to adult strains. Most children were infected with H. pylori strains lacking the cag PAI, but the presence of a complete cag PAI, in contrast to other virulence markers, was associated with more severe gastroduodenal disease.

DNA Polymorphisms in the pepA and PPE18 Genes among Clinical Strains of Mycobacterium tuberculosis: Implications for Vaccine Efficacy

ABSTRACT Tuberculosis continues to be a leading cause of death worldwide. Development of an effective vaccine against Mycobacterium tuberculosis is necessary to reduce the global burden of this disease. Mtb72F, consisting of the protein products of the pepA and PPE18 genes, is the first subunit tuberculosis vaccine to undergo phase I clinical trials. To obtain insight into the ability of Mtb72F to induce an immune response capable of recognizing different strains of M. tuberculosis, we investigated the genomic diversity of the pepA and PPE18 genes among 225 clinical strains of M. tuberculosis from two different geographical locations, Arkansas and Turkey, representing a broad range of genotypes of M. tuberculosis. A combination of single nucleotide polymorphisms (SNPs) and insertion/deletions resulting in amino acid changes in the PPE18 protein occurred in 47 (20.9%) of the 225 study strains, whereas SNPs resulted in amino acid changes in the PepA protein in 14 (6.2%) of the 225 study strains. Of the 122 Arkansas study strains and the 103 Turkey study strains, 32 (26.2%) and 15 (14.6%), respectively, had at least one genetic change leading to an alteration of the amino acid sequence of the PPE18 protein, and many of the changes occurred in regions previously reported to be potential T-cell epitopes. Thus, immunity induced by Mtb72F may not recognize a proportion of M. tuberculosis clinical strains.

Insertion- and Deletion-Associated Genetic Diversity of Mycobacterium tuberculosis Phospholipase C-Encoding Genes among 106 Clinical Isolates from Turkey

ABSTRACT Bacterial phospholipase C has been reported to play a role in the pathogenesis of many bacteria. In order to gain a better understanding of the potential role of Mycobacterium tuberculosis phospholipase C in the pathogenesis of human tuberculosis, we investigated the genetic diversity of the four M. tuberculosis phospholipase C-encoding genes (plcA, plcB, plcC, and plcD) resulting from the IS6110 insertion and associated deletion, among 106 clinical isolates obtained from Turkey, by using PCR, Southern hybridization, and DNA sequencing. Two sequenced M. tuberculosis strains, H37Rv and CDC1551, were used as the references in the comparison. Sixty-six (62.3%) of the 106 isolates had an intact plcD gene, and 40 (37.7%) showed an interruption of the gene. Of the latter 40 isolates, 19 (47.5%) had an IS6110 insertion with no associated deletion in the plcD gene, 2 (5%) had an IS6110 insertion and an associated deletion within the plcD gene, 15 (37.5%) had an IS6110 insertion in the plcD gene that was associated with a partial deletion of the plcD gene and its right forward adjacent region, and 4 (10%) had a complete deletion of the plcD gene. The proportions of the isolates with an interrupted plcA, plcB, or plcC gene were 3.8, 1.9, and 3.8%, respectively. The data indicate that there is a much higher frequency of IS6110 insertion and deletion in the plcD gene than in the plcA, plcB, and plcC genes of M. tuberculosis.

Quantitative Detection and Genotyping of Helicobacter pylori from Stool using Droplet Digital PCR Reveals Variation in Bacterial Loads that Correlates with cagA Virulence Gene Carriage

Epidemiologic studies of the carcinogenic stomach bacterium Helicobacter pylori have been limited by the lack of noninvasive detection and genotyping methods. We developed a new stool‐based method for detection, quantification, and partial genotyping of H. pylori using droplet digital PCR (ddPCR), which allows for increased sensitivity and absolute quantification by PCR partitioning.

Does the lipR gene of tubercle bacilli have a role in tuberculosis transmission and pathogenesis

Mycobacterium tuberculosis lipases, a diverse class of enzymes involved in lipid metabolism, may have an important role in tuberculosis (TB) pathogenesis. We explored the association of large sequence polymorphism (LSP) in one of the M. tuberculosis lipase-encoding genes, lipR (Rv3084), with patient characteristics using a population-based sample of clinical isolates to elucidate the potential role of lipR in TB pathogenesis. LSP in lipR was found in 104 (15.6%) of 665 isolates, of which 96% belonged to principal genetic group 3. When linkage by molecular type and epidemiologic evidence were compared, molecularly clustered cases infected with a lipR LSP isolate were more often epidemiologically linked than clustered cases infected with a lipR wild-type isolate. Further epidemiologic and functional studies are necessary to determine if the association between this lipR LSP and recent transmission we identified in this population reflects a functional role of lipR in TB transmission and pathogenesis or other unidentified mechanisms.