Leptospira interrogans is the most common cause of leptospirosis in humans and animals. Genetic analysis of L. interrogans has been severely hindered by a lack of tools for genetic manipulation. Recently we developed the mariner-based transposon Himar1 to generate the first defined mutants in L. interrogans. In this study, a total of 929 independent transposon mutants were obtained and the location of insertion determined. Of these mutants, 721 were located in the protein coding regions of 551 different genes. While sequence analysis of transposon insertion sites indicated that transposition occurred in an essentially random fashion in the genome, 25 unique transposon mutants were found to exhibit insertions into genes encoding 16S or 23S rRNAs, suggesting these genes are insertional hot spots in the L. interrogans genome. In contrast, loci containing notionally essential genes involved in lipopolysaccharide and heme biosynthesis showed few transposon insertions. The effect of gene disruption on the virulence of a selected set of defined mutants was investigated using the hamster model of leptospirosis. Two attenuated mutants with disruptions in hypothetical genes were identified, thus validating the use of transposon mutagenesis for the identification of novel virulence factors in L. interrogans. This library provides a valuable resource for the study of gene function in L. interrogans. Combined with the genome sequences of L. interrogans, this provides an opportunity to investigate genes that contribute to pathogenesis and will provide a better understanding of the biology of L. interrogans.Leptospira interrogans is a spirochete that is the main causative agent of leptospirosis. This zoonosis has emerged as a major public health problem in much of the developing world, with more than 500,000 cases of severe leptospirosis reported each year, for which the mortality rate is more than 10% (17).The genus Leptospira is composed of both saprophytic and pathogenic species. The genome sequences of two epidemic strains of L. interrogans serovars Lai and Copenhageni have been determined (20,25). More recently a human and an animal L. borgpetersenii isolate were sequenced (3), and this year, we determined the genome sequence of the saprophyte L. biflexa (22). The resulting sequences provide an invaluable source of information for identification of genetic determinants involved in the pathogenicity and environmental biology of the organism. For example, the host-adapted L. borgpetersenii genome is 16% smaller and has many more pseudogenes than the L. interrogans genome. These findings suggest that genome reduction has resulted in a reduced environmental transmission potential (3). L. interrogans has 627 genes that are absent in the L. biflexa genome, and more than 500 of these genes have unknown functions, suggesting the presence of novel virulence mechanisms (22). However, the lack of tools for L. interrogans genetics has hindered elucidation of the role of these genes in pathogenesis.Pathogenic leptospires are difficult...

ABSTRACTSpirochetes have periplasmic flagella composed of a core surrounded by a sheath. The pathogenLeptospira interroganshas fourflaB(proposed core subunit) and twoflaA(proposed sheath subunit) genes. TheflaAgenes are organized in a locus withflaA2immediately upstream offlaA1. In this study,flaA1andflaA2mutants were constructed by transposon mutagenesis. Both mutants still produced periplasmic flagella. TheflaA1mutant did not produce FlaA1 but continued to produce FlaA2 and retained normal morphology and virulence in a hamster model of infection but had reduced motility. TheflaA2mutant did not produce either the FlaA1 or the FlaA2 protein. Cells of theflaA2mutant lacked the distinctive hook-shaped ends associated withL. interrogansand lacked translational motility in liquid and semisolid media. These observations were confirmed with a second, independentflaA2mutant. TheflaA2mutant failed to cause disease in animal models of acute infection. Despite lacking FlaA proteins, the flagella of theflaA2mutant were of the same thickness as wild-type flagella, as measured by electron microscopy, and exhibited a normal flagellum sheath, indicating that FlaA proteins are not essential for the synthesis of the flagellum sheath, as observed for other spirochetes. This study shows that FlaA subunits contribute to leptospiral translational motility, cellular shape, and virulence.

Burkholderia pseudomallei is the etiologic agent of melioidosis. Many disease manifestations are associated with melioidosis, and the mechanisms causing this variation are unknown; genomic differences among strains offer one explanation. We compared the genome sequences of two strains of B. pseudomallei: the original reference strain K96243 from Thailand and strain MSHR305 from Australia. We identified a variable homologous region between the two strains. This region was previously identified in comparisons of the genome of B. pseudomallei strain K96243 with the genome of strain E264 from the closely related B. thailandensis. In that comparison, K96243 was shown to possess a horizontally acquired Yersinia-like fimbrial (YLF) gene cluster. Here, we show that the homologous genomic region in B. pseudomallei strain 305 is similar to that previously identified in B. thailandensis strain E264. We have named this region in B. pseudomallei strain 305 the B. thailandensis-like flagellum and chemotaxis (BTFC) gene cluster. We screened for these different genomic components across additional genome sequences and 571 B. pseudomallei DNA extracts obtained from regions of endemicity. These alternate genomic states define two distinct groups within B. pseudomallei: all strains contained either the BTFC gene cluster (group BTFC) or the YLF gene cluster (group YLF). These two groups have distinct geographic distributions: group BTFC is dominant in Australia, and group YLF is dominant in Thailand and elsewhere. In addition, clinical isolates are more likely to belong to group YLF, whereas environmental isolates are more likely to belong to group BTFC. These groups should be further characterized in an animal model.A gram-negative, soil-dwelling bacterium, Burkholderia pseudomallei is the causative agent of melioidosis, a tropical disease that is a significant cause of mortality and morbidity for people in Southeast Asia and northern Australia (3, 26). Many disease manifestations are associated with melioidosis (27, 28), which may be due to variation in host immune response, different modes of acquisition, or genomic differences among strains (3). B. pseudomallei is also a potential biological threat agent and is classified as a category B select agent (23) in the United States. The genome of B. pseudomallei shares a high degree of similarity with the genomes of B. thailandensis and B. mallei (15,19,29), although the former species is nonpathogenic and is thought to occur only in Thailand, and the latter species is believed to be restricted to specific host animals and is not isolated from the environment.Several recent studies have compared the whole-genome sequences of B. pseudomallei strains to those of sequenced strains of B. thailandensis and/or another closely related species, B. mallei (16,19,29). Genomic comparison of B. pseudomallei strain K96243 (15) and B. thailandensis strain E264 revealed high similarity between the two syntenic chromosomes, with most of the differences attributed to the presence of several virulence...

Leptospira interrogans is responsible for leptospirosis, a zoonosis of worldwide distribution. LipL32 is the major outer membrane protein of pathogenic leptospires, accounting for up to 75% of total outer membrane protein. In recent times LipL32 has become the focus of intense study because of its surface location, dominance in the host immune response, and conservation among pathogenic species. In this study, an lipL32 mutant was constructed in L. interrogans using transposon mutagenesis. The lipL32 mutant had normal morphology and growth rate compared to the wild type and was equally adherent to extracellular matrix. Protein composition of the cell membranes was found to be largely unaffected by the loss of LipL32, with no obvious compensatory increase in other proteins. Microarray studies found no obvious stress response or upregulation of genes that may compensate for the loss of LipL32 but did suggest an association between LipL32 and the synthesis of heme and vitamin B 12 . When hamsters were inoculated by systemic and mucosal routes, the mutant caused acute severe disease manifestations that were indistinguishable from wild-type L. interrogans infection. In the rat model of chronic infection, the LipL32 mutant colonized the renal tubules as efficiently as the wild-type strain. In conclusion, this study showed that LipL32 does not play a role in either the acute or chronic models of infection. Considering the abundance and conservation of LipL32 among all pathogenic Leptospira spp. and its absence in saprophytic Leptospira, this finding is remarkable. The role of this protein in leptospiral biology and pathogenesis thus remains elusive.

Background Burkholderia pseudomallei, a Gram-negative bacterium that causes melioidosis, was reported to produce biofilm. As the disease causes high relapse rate when compared to other bacterial infections, it therefore might be due to the reactivation of the biofilm forming bacteria which also provided resistance to antimicrobial agents. However, the mechanism on how biofilm can provide tolerance to antimicrobials is still unclear.Methodology/Principal FindingsThe change in resistance of B. pseudomallei to doxycycline, ceftazidime, imipenem, and trimethoprim/sulfamethoxazole during biofilm formation were measured as minimum biofilm elimination concentration (MBEC) in 50 soil and clinical isolates and also in capsule, flagellin, LPS and biofilm mutants. Almost all planktonic isolates were susceptible to all agents studied. In contrast, when they were grown in the condition that induced biofilm formation, they were markedly resistant to all antimicrobial agents even though the amount of biofilm production was not the same. The capsule and O-side chains of LPS mutants had no effect on biofilm formation whereas the flagellin-defective mutant markedly reduced in biofilm production. No alteration of LPS profiles was observed when susceptible form was changed to resistance. The higher amount of N-acyl homoserine lactones (AHLs) was detected in the high biofilm-producing isolates. Interestingly, the biofilm mutant which produced a very low amount of biofilm and was sensitive to antimicrobial agents significantly resisted those agents when grown in biofilm inducing condition.Conclusions/SignificanceThe possible drug resistance mechanism of biofilm mutants and other isolates is not by having biofilm but rather from some factors that up-regulated when biofilm formation genes were stimulated. The understanding of genes related to this situation may lead us to prevent B. pseudomallei biofilms leading to the relapse of melioidosis.