A \u3cem\u3eMycobacterium ulcerans\u3c/em\u3e Toxin, Mycolactone, Causes Apoptosis in Guinea Pig Ulcers and Tissue Culture Cells

Mycobacterium ulcerans is the causative agent of Buruli ulcer, a tropical ulcerative skin disease. One of the most intriguing aspects of this disease is the presence of extensive tissue damage in the absence of an acute inflammatory response. We recently purified and characterized a macrolide toxin, mycolactone, from M. ulcerans. Injection of this molecule into guinea pig skin reproduced cell death and lack of acute inflammatory response similar to that seen following the injection of viable bacteria. We also showed that mycolactone causes a cytopathic effect on mouse fibroblast L929 cells that is characterized by cytoskeletal rearrangements and growth arrest within 48 h. However, these results could not account for the extensive cell death which occurs in Buruli ulcer. The results presented here demonstrate that L929 and J774 mouse macrophage cells die via apoptosis after 3 to 5 days of exposure to mycolactone. Treatment of cells with a pan-caspase inhibitor can inhibit mycolactone-induced apoptosis. We demonstrate that injection of mycolactone into guinea pig skin results in cell death via apoptosis and that the extent of apoptosis increases as the lesion progresses. These results may help to explain why tissue damage in Buruli ulcer is not accompanied by an acute inflammatory response

Host Restriction Phenotypes of \u3cem\u3eSalmonella typhi\u3c/em\u3e and \u3cem\u3eSalmonella gallinarum\u3c/em\u3e

Salmonella typhi and Salmonella gallinarum phenotypes correlated with mouse host restriction have been identified by using in vitro and in vivo systems. S. typhi is capable of entering the murine intestinal epithelium via M cells, as is Salmonella typhimurium, which causes systemic infection in the mouse. But, unlike S. typhimurium, S. typhi does not destroy the epithelium and is cleared from the Peyer’s patches soon after M-cell entry. S. gallinarum appears to be incapable of entering the murine Peyer’s patch epithelium. Our in vitro evidence suggests that S. gallinarum is taken up in murine phagocytic cells by a mechanism different from that of S. typhimurium. S. typhimurium is taken up at a higher frequency and is maintained at higher viable counts throughout a 24-h time course in a murine macrophage-like cell line than are S. gallinarum and S. typhi

Genetic Basis of Virulence Attenuation Revealed by Comparative Genomic Analysis of Mycobacterium tuberculosis Strain H37Ra versus H37Rv

Tuberculosis, caused by Mycobacterium tuberculosis, remains a leading infectious disease despite the availability of chemotherapy and BCG vaccine. The commonly used avirulent M. tuberculosis strain H37Ra was derived from virulent strain H37 in 1935 but the basis of virulence attenuation has remained obscure despite numerous studies. We determined the complete genomic sequence of H37Ra ATCC25177 and compared that with its virulent counterpart H37Rv and a clinical isolate CDC1551. The H37Ra genome is highly similar to that of H37Rv with respect to gene content and order but is 8,445 bp larger as a result of 53 insertions and 21 deletions in H37Ra relative to H37Rv. Variations in repetitive sequences such as IS6110 and PE/PPE/PE-PGRS family genes are responsible for most of the gross genetic changes. A total of 198 single nucleotide variations (SNVs) that are different between H37Ra and H37Rv were identified, yet 119 of them are identical between H37Ra and CDC1551 and 3 are due to H37Rv strain variation, leaving only 76 H37Ra-specific SNVs that affect only 32 genes. The biological impact of missense mutations in protein coding sequences was analyzed in silico while nucleotide variations in potential promoter regions of several important genes were verified by quantitative RT-PCR. Mutations affecting transcription factors and/or global metabolic regulations related to in vitro survival under aging stress, and mutations affecting cell envelope, primary metabolism, in vivo growth as well as variations in the PE/PPE/PE-PGRS family genes, may underlie the basis of virulence attenuation. These findings have implications not only for improved understanding of pathogenesis of M. tuberculosis but also for development of new vaccines and new therapeutic agents

Differentially Evolved Genes of Salmonella Pathogenicity Islands: Insights into the Mechanism of Host Specificity in Salmonella

BACKGROUND: The species Salmonella enterica (S. enterica) includes many serovars that cause disease in avian and mammalian hosts. These serovars differ greatly in their host range and their degree of host adaptation. The host specificity of S. enterica serovars appears to be a complex phenomenon governed by multiple factors acting at different stages of the infection process, which makes identification of the cause/s of host specificity solely by experimental methods difficult. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we have employed a molecular evolution and phylogenetics based approach to identify genes that might play important roles in conferring host specificity to different serovars of S. enterica. These genes are 'differentially evolved' in different S. enterica serovars. This list of 'differentially evolved' genes includes genes that encode translocon proteins (SipD, SseC and SseD) of both Salmonella pathogenicity islands 1 and 2 encoded type three secretion systems, sptP, which encodes an effector protein that inhibits the mitogen-activated protein kinase pathway of the host cell, and genes which encode effector proteins (SseF and SifA) that are important in placing the Salmonella-containing vacuole in a juxtanuclear position. CONCLUSIONS/SIGNIFICANCE: Analysis of known functions of these 'differentially evolved genes' indicates that the products of these genes directly interact with the host cell and manipulate its functions and thereby confer host specificity, at least in part, to different serovars of S. enterica that are considered in this study

Evolution of Salmonella enterica Virulence via Point Mutations in the Fimbrial Adhesin

Whereas the majority of pathogenic Salmonella serovars are capable of infecting many different animal species, typically producing a self-limited gastroenteritis, serovars with narrow host-specificity exhibit increased virulence and their infections frequently result in fatal systemic diseases. In our study, a genetic and functional analysis of the mannose-specific type 1 fimbrial adhesin FimH from a variety of serovars of Salmonella enterica revealed that specific mutant variants of FimH are common in host-adapted (systemically invasive) serovars. We have found that while the low-binding shear-dependent phenotype of the adhesin is preserved in broad host-range (usually systemically non-invasive) Salmonella, the majority of host-adapted serovars express FimH variants with one of two alternative phenotypes: a significantly increased binding to mannose (as in S. Typhi, S. Paratyphi C, S. Dublin and some isolates of S. Choleraesuis), or complete loss of the mannose-binding activity (as in S. Paratyphi B, S. Choleraesuis and S. Gallinarum). The functional diversification of FimH in host-adapted Salmonella results from recently acquired structural mutations. Many of the mutations are of a convergent nature indicative of strong positive selection. The high-binding phenotype of FimH that leads to increased bacterial adhesiveness to and invasiveness of epithelial cells and macrophages usually precedes acquisition of the non-binding phenotype. Collectively these observations suggest that activation or inactivation of mannose-specific adhesive properties in different systemically invasive serovars of Salmonella reflects their dynamic trajectories of adaptation to a life style in specific hosts. In conclusion, our study demonstrates that point mutations are the target of positive selection and, in addition to horizontal gene transfer and genome degradation events, can contribute to the differential pathoadaptive evolution of Salmonella

Site-specific integration of mycobacteriophage L5: integration-proficient vectors for Mycobacterium smegmatis, Mycobacterium tuberculosis, and bacille Calmette-Guérin.

Mycobacteriophage L5, a temperate phage of mycobacteria, integrates site-specifically into the Mycobacterium smegmatis chromosome. We have identified the int gene and attP site of L5, characterized the chromosomal attachment site (attB), and constructed plasmid vectors that efficiently transform M. smegmatis through stable site-specific integration of the plasmid into the bacterial genome. These integration-proficient plasmids also efficiently transform slow-growing mycobacteria such as the pathogen Mycobacterium tuberculosis and the vaccine strain bacille Calmette-Guérin (BCG). The ability to easily generate stable recombinants in these slow-growing mycobacteria without the requirement for continual selection is of particular importance for the construction of recombinant BCG vaccines and for the isolation and characterization of mycobacterial pathogenic determinants in animal model systems. Integration vectors of this type should be of general use in a number of additional bacterial systems where temperate phages have been identified

Use of in vivo complementation in Mycobacterium tuberculosis to identify a genomic fragment associated with virulence

Novel molecular tools and genetic methods were developed to isolate genomic fragments of Mycobacterium tuberculosis that may be associated with virulence. We sought to restore virulence, a characteristic of M. tuberculosis that is correlated with growth rate in mouse spleen and lung tissue, to the avirulent strain H37Ra by complementation. A representative library of the virulent M. tuberculosis strain H37Rv was constructed and transformed into H37Ra. Enrichment for individual faster-growing recombinants was achieved by passage of pools of H37Ra transformants harboring the H37Rv library through mice. A molecular strategy was devised to isolate and clone the H37Rv genomic DNA fragment ivg, which conferred a more rapid in vivo growth rate to H37Ra