Rasana W. Sermswan

Major Surface Protein LipL32 Is Not Required for Either Acute or Chronic Infection with Leptospira interrogans

ABSTRACT 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 B12. 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.

Genome-wide transposon mutagenesis in pathogenic Leptospira species.

ABSTRACT 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.

Development of a PCR-based method for the detection of Opisthorchis viverrini in experimentally infected hamsters

Opisthorchis viverrini infection is an endemic disease that causes a serious public health problem in southeast Asia, especially in northeast Thailand. We have developed a PCR method using a pair of primers named OV-6F/OV-6R for detecting O. viverrini eggs in stool samples and compared it with Stolls egg-count method. The primers were designed based on the pOV-A6 specific DNA probe sequence which gave a 330 base pair product. The PCR method can detect a single egg in artificially inoculated faeces or as little as 2 x 10(-17) ng of O. viverrini genomic DNA. The method gave 100% sensitivity in all hamster groups except in animals exposed to the lowest intensity of infection (1 metacercaria/hamster). In the first month of infection, the PCR method was more sensitive than using the egg-count method in all infected groups especially in the light infections. The PCR method was also successfully used in monitoring a therapeutic study. Since the PCR method showed no cross-reaction with Heterophyid flukes, it can be useful for specific identification of O. viverrini eggs in stool samples without the risk of false positives. It also has great potential for application in clinical epidemiological studies.

FlaA Proteins in Leptospira interrogans Are Essential for Motility and Virulence but Are Not Required for Formation of the Flagellum Sheath

ABSTRACT Spirochetes have periplasmic flagella composed of a core surrounded by a sheath. The pathogen Leptospira interrogans has four flaB (proposed core subunit) and two flaA (proposed sheath subunit) genes. The flaA genes are organized in a locus with flaA2 immediately upstream of flaA1. In this study, flaA1 and flaA2 mutants were constructed by transposon mutagenesis. Both mutants still produced periplasmic flagella. The flaA1 mutant 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. The flaA2 mutant did not produce either the FlaA1 or the FlaA2 protein. Cells of the flaA2 mutant lacked the distinctive hook-shaped ends associated with L. interrogans and lacked translational motility in liquid and semisolid media. These observations were confirmed with a second, independent flaA2 mutant. The flaA2 mutant failed to cause disease in animal models of acute infection. Despite lacking FlaA proteins, the flagella of the flaA2 mutant 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.

A Horizontal Gene Transfer Event Defines Two Distinct Groups within Burkholderia pseudomallei That Have Dissimilar Geographic Distributions

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.

Leptospira interrogans requires heme oxygenase for disease pathogenesis

We recently characterised the Leptospira interrogans heme oxygenase (hemO) gene and showed that HemO was required for growth with hemoglobin as the sole iron source. Here we investigated the role of HemO in pathogenesis. Hamsters inoculated with the hemO mutant showed 83% survival, compared with 33% for a control mutant (intergenic transposon insertion). Lung pathology was consistent with survival data, showing that HemO contributes significantly to pathogenesis and heme is a major in vivo iron source for L. interrogans. This is only the second defined, attenuated mutant in pathogenic Leptospira and the first to define function of the mutated gene.

Soil physicochemical properties related to the presence of Burkholderia pseudomallei.

The incidence of Burkholderia pseudomallei in the soil from north-east Thailand is estimated to be 20-fold higher than that from central Thailand, and is associated with a 10-fold higher incidence of melioidosis in the region than in central Thailand. This study investigated the presence of B. pseudomallei in relation to the physicochemical properties of soil from Khon Kaen province, north-east Thailand. Thirteen districts (54.2%) were positive for B. pseudomallei. From a selected district, B. pseudomallei was cultured from 19 of 50 sites (38%). The soil in this area was predominantly sandy. From the positive sites, the organism was found mainly at a depth of 30 cm (43/68, 63% of isolates) and was significantly associated with certain soil physicochemical parameters, including a pH of 5.0-6.0, a moisture content >10%, and higher chemical oxygen demand and total nitrogen than negative sites (P<0.05). Burkholderia pseudomallei is unevenly distributed in this area, with the pH of the soil being the major determinant of the presence of the organism. The sandy soil type of north-east Thailand can support the survival of B. pseudomallei and allow it to move freely with water flow, and thus readily come in contact with people during the rainy season.

Mutations affecting Leptospira interrogans lipopolysaccharide attenuate virulence

Leptospira interrogans is the causative agent of leptospirosis. Lipopolysaccharide (LPS) is the major outer membrane component of L. interrogans. It is the dominant antigen recognized during infection and the basis for serological classification. The structure of LPS and its role in pathogenesis are unknown. We describe two defined mutants of L. interrogans serovar Manilae with transposon insertions in the LPS locus. Mutant M895 was disrupted in gene la1641 encoding a protein with no known homologues. M1352 was disrupted in a gene unique to serovar Manilae also encoding a protein of unknown function. M895 produced truncated LPS while M1352 showed little or no change in LPS molecular mass. Both mutants showed altered agglutination titres against rabbit antiserum and against a panel of LPS‐specific monoclonal antibodies. The mutants were severely attenuated in virulence via the intraperitoneal route of infection, and were cleared from the host animal by 3 days after infection. M895 was also highly attenuated via the mucosal infection route. Resistance to complement in human serum was unaltered for both mutants. While complementation of mutants was not possible, the attenuation of two independently derived LPS mutants demonstrates for the first time that LPS plays an essential role leptospiral virulence.

Leptospira interrogans Catalase Is Required for Resistance to H2O2 and for Virulence

ABSTRACT Pathogenic Leptospira spp. are likely to encounter higher concentrations of reactive oxygen species induced by the host innate immune response. In this study, we characterized Leptospira interrogans catalase (KatE), the only annotated catalase found within pathogenic Leptospira species, by assessing its role in resistance to H2O2-induced oxidative stress and during infection in hamsters. Pathogenic L. interrogans bacteria had a 50-fold-higher survival rate under H2O2-induced oxidative stress than did saprophytic L. biflexa bacteria, and this was predominantly catalase dependent. We also characterized KatE, the only annotated catalase found within pathogenic Leptospira species. Catalase assays performed with recombinant KatE confirmed specific catalase activity, while protein fractionation experiments localized KatE to the bacterial periplasmic space. The insertional inactivation of katE in pathogenic Leptospira bacteria drastically diminished leptospiral viability in the presence of extracellular H2O2 and reduced virulence in an acute-infection model. Combined, these results suggest that L. interrogans KatE confers in vivo resistance to reactive oxygen species induced by the host innate immune response.

Recombinant LipL32 and LigA from Leptospira are unable to stimulate protective immunity against leptospirosis in the hamster model

The major antigenic component of pathogenic Leptospira spp. is lipopolysaccharide (LPS). However, due to the specificity of the immune response generated towards LPS and the diversity in leptospiral LPS carbohydrate structure, current commercial vaccines stimulate protection only against homologous or closely related serovars. Vaccines that confer heterologous protection would enhance protection in vaccinated animals and reduce transmission to humans. Several studies have investigated the potential of various leptospiral outer membrane proteins to stimulate protective immunity against pathogenic Leptospira species. These include the surface-exposed lipoproteins LipL32 and LigA. However, consistent protection from infection has proved difficult to reproduce. In this study we assessed the protective capacity of recombinant LipL32, the six carboxy-terminal unique Ig-like repeat domains of LigA (LigANI) and a LipL32-LigANI fusion protein in hamsters against infection with Leptospira interrogans serovar Manilae. Despite all of the proteins eliciting antibody responses, none of the hamsters was protected against infection.