The spirochete FlaA periplasmic flagellar sheath protein impacts flagellar helicity

Spirochete periplasmic flagella (PFs), including those from Brachyspira (Serpulina), Spirochaeta, Treponema, and Leptospira spp., have a unique structure. In most spirochete species, the periplasmic flagellar filaments consist of a core of at least three proteins (FlaB1, FlaB2, and FlaB3) and a sheath protein (FlaA). Each of these proteins is encoded by a separate gene. Using Brachyspira hyodysenteriae as a model system for analyzing PF function by allelic exchange mutagenesis, we analyzed purified PFs from previously constructed flaA::cat, flaA::kan, and flaB1::kan mutants and newly constructed flaB2::cat and flaB3::cat mutants. We investigated whether any of these mutants had a loss of motility and altered PF structure. As formerly found with flaA::cat, flaA::kan, and flaB1::kan mutants, flaB2::cat and flaB3::cat mutants were still motile, but all were less motile than the wild-type strain, using a swarm-plate assay. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis indicated that each mutation resulted in the specific loss of the cognate gene product in the assembled purified PFs. Consistent with these results, Northern blot analysis indicated that each flagellar filament gene was monocistronic. In contrast to previous results that analyzed PFs attached to disrupted cells, purified PFs from a flaA::cat mutant were significantly thinner (19.6 nm) than those of the wild-type strain and flaB1::kan, flaB2::cat, and flaB3::cat mutants (24 to 25 nm). These results provide supportive genetic evidence that FlaA forms a sheath around the FlaB core. Using high-magnification dark-field microscopy, we also found that flaA::cat and flaA::kan mutants produced PFs with a smaller helix pitch and helix diameter compared to the wild-type strain and flaB mutants. These results indicate that the interaction of FlaA with the FlaB core impacts periplasmic flagellar helical morphology.     

First evidence for gene replacement in Leptospira spp. Inactivation of L. biflexa flaB results in non-motile mutants deficient in endoflagella

Leptospira spp. offer many advantages as model bacteria for the study of spirochaetes. However, homologous recombination between introduced DNA and the corresponding chromosomal loci has never been demonstrated. A unique feature of spirochaetes is the presence of endoflagella between the outer membrane sheath and the cell cylinder. We chose the flaB flagellin gene, constituting the flagellar core, as a target for gene inactivation in the saprophyte Leptospira biflexa. The amino acid sequence of the FlaB protein of L. biflexa was most similar to those of spirochaetes Brachyspira hyodysenteriae (agent of swine dysentery), Leptospira interrogans (agent of leptospirosis) and Treponema pallidum (agent of syphilis). A suicide vector containing the L. biflexa flaB gene disrupted by a kanamycin marker was UV irradiated or alkali denatured before electroporation. This methodology allowed the selection of many kanamycin-resistant colonies resulting from single and double cross-over events at the flaB locus. The double recombinant mutants are non-motile, as visualized in both liquid and semi-solid media. In addition, a flaB mutant selected for further analysis was shown to be deficient in endoflagella by electron microscopy. However, most of the transformants had resulted from a single homologous recombination event, giving rise to the integration of the suicide vector. We evaluated the effect of the sacB and rpsL genes in L. biflexa as potential counterselectable markers for allelic exchange, and then used the rpsL system for the positive selection of flaB double recombinants in a streptomycin-resistant strain. Like the flaB mutant studied above, the Strr double cross-over mutant was non-motile and deficient in endoflagella. Our results demonstrate that FlaB is involved in flagella assembly and motility. They also show the feasibility of performing allelic replacement in Leptospira spp. by homologous recombination.     

Simultaneous detection and differentiation of pathogenic and nonpathogenic Leptospira spp. by multiplex real-time PCR (TaqMan) assay

Leptospirosis, caused by pathogenic Leptospira, is one of the most important zoonoses in the world. Several molecular techniques have been developed for detection and differentiation between pathogenic and saprophytic Leptospira spp. The aim of this study was to develop a rapid and simple assay for specific detection and differentiation of pathogenic Leptospira spp. by multiplex real-time PCR (TaqMan) assay using primers and probes targeting Leptospira genus specific 16S ribosomal RNA gene, the pathogen specific lig A/B genes and nonpathogen Leptospira biflexa specific 23S ribosomal RNA gene. Sixteen reference strains of Leptospira spp. including pathogenic and nonpathogenic and ten other negative control bacterial strains were used in the study. While the 16S primers amplified target from both pathogenic and non-pathogenic leptospires, the ligA/B and the 23S primers amplified target DNA from pathogenic and non-pathogenic leptospires, respectively. The multiplex real-time PCR (TaqMan) assay detection limit, that is, the sensitivity was found approximately 1 x 10(2) cells/ml for ligA/B gene and 23S ribosomal RNA gene, and 10 cells/ml 16S ribosomal RNA. The reaction efficiencies were 83-105% with decision coefficients of more than 0.99 in all multiplex assays. The multiplex real-time PCR (TaqMan) assay yielded negative results with the ten other control bacteria. In conclusion, the developed multiplex real-time PCR (TaqMan) assay is highly useful for early diagnosis and differentiation between pathogenic and non-pathogenic leptospires in a reaction tube as having high sensitivity and specificity.     

Molecular analysis of a Leptospira borgpetersenii gene encoding an endoflagellar subunit protein

A flagellin gene, flaB, from Leptospira borgpetersenii (formerly L. interrogans) serovar hardjo was cloned and expressed in Escherichia coli. Expression of the 32 kDa FlaB protein was dependent upon the lacZ promoter from pUC18. Nucleotide sequence data showed an open reading frame encoding 283 amino acid residues, corresponding to a protein of molecular mass 31.3 kDa. The G + C content of the flaB gene was 54.7 mol%. Comparison of the deduced FlaB amino acid sequence with flagellins from other bacteria revealed a high level of identity with the Treponema pallidum FlaB proteins.     

InvA protein is a Nudix hydrolase required for infection by pathogenic Leptospira in cell lines and animals

Leptospirosis caused by pathogenic species of the genus Leptospira is a re-emerging zoonotic disease, which affects a wide variety of host species and is transmitted by contaminated water. The genomes of several pathogenic Leptospira species contain a gene named invA, which contains a Nudix domain. However, the function of this gene has never been characterized. Here, we demonstrated that the invA gene was highly conserved in protein sequence and present in all tested pathogenic Leptospira species. The recombinant InvA protein of pathogenic L. interrogans strain Lai hydrolyzed several specific dinucleoside oligophosphate substrates, reflecting the enzymatic activity of Nudix in Leptospira species. Pathogenic leptospires did not express this protein in media but temporarily expressed it at early stages (within 60 min) of infection of macrophages and nephric epithelial cells. Comparing with the wild type, the invA-deficient mutant displayed much lower infectivity and a significantly reduced survival rate in macrophages and nephric epithelial cells. Moreover, the invA-deficient leptospires presented an attenuated virulence in hamsters, caused mild histopathological damage, and were transmitted in lower numbers in the urine, compared with the wild-type strain. The invA revertant, made by complementing the invA-deficient mutant with the invA gene, reacquired virulence similar to the wild type in vitro and in vivo. The LD(50) in hamsters was 1000-fold higher for the invA-deficient mutant than for the invA revertant and wild type. These results demonstrate that the InvA protein is a Nudix hydrolase, and the invA gene is essential for virulence in pathogenic Leptospira species.     

Leptospira species categorized by arbitrarily primed polymerase chain reaction (PCR) and by mapped restriction polymorphisms in PCR-amplified rRNA genes.

Reference strains from 48 selected serovars representing eight species of Leptospira were examined by two polymerase chain reaction (PCR)-based strategies. First, mapped restriction site polymorphisms (MRSP) were examined in PCR products from portions of rrs (16S rRNA gene) and rrl (23S rRNA gene). Twenty MRSP and 2 length polymorphisms were used to group reference strains into 16 MRSP profiles. Species assignments were consistent with those obtained by a second method, genomic fingerprinting with arbitrarily primed PCR, in which strains within a species were characterized by many shared arbitrarily primed PCR products. The results of both of these methods were in general agreement with those of previous studies that used DNA-DNA relatedness and confirmed the high level of divergence among the recognized species of Leptospira. However, Leptospira meyeri serovar ranarum and evansi strains were indistinguishable from some strains of Leptospira interrogans sensu stricto. Intervening sequences of about 485 to 740 bp were located near base 1230 in rrl of some strains.     

Brachyspira (Serpulina) hyodysenteriae gyrB mutants and interstrain transfer of coumermycin A(1) resistance

To further develop genetic techniques for the enteropathogen Brachyspira hyodysenteriae, the gyrB gene of this spirochete was isolated from a lambdaZAPII library of strain B204 genomic DNA and sequenced. The putative protein encoded by this gene exhibited up to 55% amino acid sequence identity with GyrB proteins of various bacterial species, including other spirochetes. B. hyodysenteriae coumermycin A(1)-resistant (Cn(r)) mutant strains, both spontaneous and UV induced, were isolated by plating B204 cells onto Trypticase soy blood agar plates containing 0.5 microg of coumermycin A(1)/ml. The coumermycin A(1) MICs were 25 to 100 microg/ml for the resistant strains and 0.1 to 0.25 microg/ml for strain B204. Four Cn(r) strains had single nucleotide changes in their gyrB genes, corresponding to GyrB amino acid changes of Gly(78) to Ser (two strains), Gly(78) to Cys, and Thr(166) to Ala. When Cn(r) strain 435A (Gly(78) to Ser) and Cm(r) Km(r) strain SH (DeltaflaA1::cat Deltanox::kan) were cultured together in brain heart infusion broth containing 10% (vol/vol) heat-treated (56 degrees C, 30 min) calf serum, cells resistant to chloramphenicol, coumermycin A(1), and kanamycin could be isolated from the cocultures after overnight incubation, but such cells could not be isolated from monocultures of either strain. Seven Cn(r) Km(r) Cm(r) strains were tested and were determined to have resistance genotypes of both strain 435A and strain SH. Cn(r) Km(r) Cm(r) cells could not be isolated when antiserum to the bacteriophage-like agent VSH-1 was added to cocultures, and the numbers of resistant cells increased fivefold when mitomycin C, an inducer of VSH-1 production, was added. These results indicate that coumermycin resistance associated with a gyrB mutation is a useful selection marker for monitoring gene exchange between B. hyodysenteriae cells. Gene transfer readily occurs between B. hyodysenteriae cells in broth culture, a finding with practical importance. VSH-1 is the likely mechanism for gene transfer.     

致病钩体表层膜蛋白新基因（Lslp）的克隆表达及免疫原性研究

克隆表达钩端螺旋体表层膜蛋白新基因Lslp并分析表达产物的免疫原性。根据前期研究得到的致病钩体新基因Lslp(GenBankAF32 5 80 7)的序列设计引物 ,在 6株致病钩体中扩增Lslp基因并测序。以BamHⅠ酶切Lslp和pGEX 1 λT ,构建重组质粒并用酶切和PCR鉴定 ,进一步在大肠杆菌中诱导表达 ,并进行免疫印迹分析 ;纯化表达产物免疫家兔 ,ELISA检测血清抗体滴度。结果显示Lslp在 6株致病钩体中均能扩增出相应片段 ,且序列同源性达到99 6 % ;构建高效原核表达重组质粒pGST LslP ,经IPTG诱导在大肠杆菌中可表达出 6 6kDGST融合蛋白 ,并能与全钩抗血清发生免疫印迹反应 ;将上述融合蛋白免疫新西兰大白兔产生 1 :5 1 2 0高滴度的IgG抗体。研究结果提示致病钩体膜蛋白新基因Lslp可在大肠杆菌进行高效表达 ,表达产物能被全钩抗血清识别 ,为研究钩体的致病机制和筛选保护性抗原提供了基础     

A PCR assay for the detection of Campylobacter jejuni and Campylobacter coli in water

A PCR assay has been developed for the detection of Campylobacter jejuni and Camp. coli in water samples. The sample is filtered through a membrane which is subjected to sonication to release the impacted cells. After removal of the filter from the cell suspension and a freeze/thaw cell lysis step, a semi-nested PCR is carried out on the filtrate using the primers CF02, CF03 and CF04 ( Camp. jejuni fla and flaB gene sequences). Incorporation of a sonication stage allows removal of the filter membrane since they have been shown to inhibit the PCR. In experiments with spiked water samples (20 ml) a theoretical sensitivity of 10–20 Campylobacter cells ml^-1 was achieved. Using a sample volume of 100 ml this sensitivity can be increased to approximately 2 Campylobacter cells ml^-1.     

Development of species-specific PCR primer sets for the detection of Leptospira

Spirochetes of the genus Leptospira infect animals and humans and are the causative agents for the emerging infectious disease leptospirosis. Rapid and simple assays for the identification of individual Leptospira species are currently not available. For identification of individual Leptospira species, PCR primers that detect the ompL1 gene sequence for the majority of pathogenic leptospires were developed in this study. The primer pairs detect Leptospira interrogans, Leptospira borgpetersenii, Leptospira kirschneri, Leptospira santarosai, Leptospira weilii and Leptospira noguchii, without cross-reacting with other Leptospira species. The development of the primers revealed a divergence of the ompL1 gene within L. interrogans, splitting this species into two separate groups. The species-specific primers will be especially useful in epidemiological studies and disease outbreak investigations for the detection of Leptospira species in human, animal and environmental samples.     

First record of Leptospira borgpetersenii isolation in the Amami Islands, Japan

In 2003, a Leptospira survey was performed on Yoroshima Island of the Amami Islands located in the southwestern part of Japan. Seven Leptospira strains were isolated from the field rat Rattus rattus, which were identified as L. borgpetersenii by flaB sequencing, 16S rDNA sequencing and gyrB sequencing, and serovar Javanica was determined by a microscopic agglutination test. NotI-long restriction fragment analysis indicated that these isolates were genetically indistinguishable from an isolate from the Okinawa Islands. The present results suggest that L. borgpetersenii is migrating into the Amami Islands in Japan.     

Comparative ultrastructural and functional studies of Helicobacter pylori and Helicobacter mustelae flagellin mutants: both flagellin subunits, FlaA and FlaB, are necessary for full motility in Helicobacter species.

Helicobacter mustelae causes chronic gastritis and ulcer disease in ferrets. It is therefore considered an important animal model of human Helicobacter pylori infection. High motility even in a viscous environment is one of the common virulence determinants of Helicobacter species. Their sheathed flagella contain a complex filament that is composed of two distinctly different flagellin subunits, FlaA and FlaB, that are coexpressed in different amounts. Here, we report the cloning and sequence determination of the flaA gene of H. mustelae NCTC12032 from a PCR amplification product. The FlaA protein has a calculated molecular mass of 53 kDa and is 73% homologous to the H. pylori FlaA subunit. Isogenic flaA and flaB mutants of H. mustelae F1 were constructed by means of reverse genetics. A method was established to generate double mutants (flaA flaB) of H. mustelae F1 as well as H. pylori N6. Genotypes, motility properties, and morphologies of the H. mustelae flagellin mutants were determined and compared with those of the H. pylori flaA and flaB mutants described previously. The flagellar organizations of the two Helicobacter species proved to be highly similar. When the flaB genes were disrupted, motility decreased by 30 to 40%. flaA mutants retained weak motility by comparison with strains that were devoid of both flagellin subunits. Weakly positive motility tests of the flaA mutants correlated with the existence of short truncated flagella. In H. mustelae, lateral as well as polar flagella were present in the truncated form. flaA flaB double mutants were completely nonmotile and lacked any form of flagella. These results show that the presence of both flagellin subunits is necessary for complete motility of Helicobacter species. The importance of this flagellar organization for the ability of the bacteria to colonize the gastric mucosa and to persist in the gastric mucus remains to be proven.     

Identification and partial characterization of a novel hemolysin from Leptospira interrogans serovar lai

It has been suggested that leptospiral hemolysins are important in the virulence and pathogenesis of leptospirosis. We have isolated an Escherichia coli clone carrying the 7.8kb DNA insert from a genomic library of Leptospira interrogans serovar lai by plaque hybridization using a sequence derived from the sphingomyelinase C gene (sphA) of L. borgpetersenii. The clone showed a clear beta-hemolytic zone on sheep blood agar and high hemolytic activities on both human and sheep erythrocytes in liquid assays. The clone carried at least two genes responsible for the hemolytic activities, encoded by two open reading frames of 1662 and 816 nucleotides, which are named sphH and hap-1 (hemolysis associated protein-1), respectively. The SphH showed 75% homology to the SphA at the amino acid level, and the Hap-1 showed no significant homology in major databases. Interestingly, however, E. coli cells harboring sphH did not show sphingomyelinase or phospholipase activities. Moreover, SphH-mediated hemolysis was osmotically protected by polyethylene glycol 5000, suggesting that the hemolysis is likely to be caused by pore formation on the membrane. The SphH was successfully expressed in E. coli as a histidine (His)-SphH fusion protein. Both sphH and hap-1 were highly conserved among the Leptospira species, except for the absence of sphH in non-pathogenic L. biflexa serovar patoc. We concluded that the SphH is a novel hemolysin of a pathogenic Leptospira species, which may be a putative pore-forming protein.     

Polymerase chain reaction assay specific for pathogenic Leptospira based on the gene hap1 encoding the hemolysis-associated protein-1

In this study, we used Southern hybridization of genomic DNA with the integral hap1 gene as a probe to show that this gene is only present in pathogenic Leptospira strains. We then selected PCR primers based on the hap1 gene, and tested them on several Leptospira strains and biological samples. Specific amplification was obtained for all pathogenic strains tested. Negative PCR results were observed with all saprophytic leptospire strains used as well as with other spirochetes and bacteria commonly found in biological samples. The results of direct PCR performed on biological samples, such as blood, urine or kidneys correlated with the results obtained with the classical Leptospira tests (culture and MAT). A PCR assay based on this gene would be a very useful tool for the rapid, sensitive and specific identification of pathogenic leptospires in samples for diagnosis or epidemiological survey.     

flaB gene as a molecular marker for distinct identification of Borrelia species in environmental samples by the PCR-restriction fragment length polymorphism method

A new protocol employing nested PCR-restriction fragment length polymorphism (RFLP) based on the flaB gene and two restriction enzymes was worked out. This protocol allows the identification of all Borrelia species transmitted by Ixodes ricinus in Europe, including Borrelia miyamotoi and 3 genetic variants of B. garinii. A dendrogram of flaB sequence similarity was in accordance with RFLP variants.     

Complementation of a Nonmotile flaB Mutant of Borrelia burgdorferi by Chromosomal Integration of a Plasmid Containing a Wild-Type flaB Allele

With the recent identification of antibiotic resistance phenotypes, the use of reporter genes, the isolation of null mutants by insertional inactivation, and the development of extrachromosomal cloning vectors, genetic analysis of Borrelia burgdorferi is becoming a reality. A previously described nonmotile, rod-shaped, kanamycin-resistant B. burgdorferi flaB::Km null mutant was complemented by electroporation with the erythromycin resistance plasmid pED3 (a pGK12 derivative) containing the wild-type flaB sequence and 366 bp upstream from its initiation codon. The resulting MS17 clone possessed erythromycin and kanamycin resistance, flat-wave morphology, and microscopic and macroscopic motility. Several other electroporations with plasmids containing wild-type flaB and various lengths (198, 366, or 762 bp) of sequence upstream from the flaB gene starting codon did not lead to functional restoration of the nonmotile flaB null mutant. DNA hybridization, PCR analysis, and sequencing indicated that the wild-type flaB gene in nonmotile clones was present in the introduced extrachromosomal plasmids, while the motile MS17 clone was a merodiploid containing single tandem chromosomal copies of mutated flaB::Km and wild-type flaB with a 366-bp sequence upstream from its starting codon. Complementation was thus achieved only when wild-type flaB was inserted into the borrelial chromosome. Several possible mechanisms for the failure of complementation for extrachromosomally located flaB are discussed.     

Genomic rearrangements in the flagellin genes of Proteus mirabilis

Molecular analyses have revealed that Proteus mirabilis possesses two genes, flaA and flaB, that are homologous to each other and to flagellin genes of many other species. Both swimmer and swarmer cells transcribe flaA, but not flaB. FlaA- mutants are non-motile and do not differentiate showing the essential role of flaA in swarmer cell differentiation and behaviour. At a low frequency, motile, differentiation-proficient revertants have been found in FlaA-populations. These revertants produce an antigenically and biochemically distinct flagellin protein. The revertant flagellin is the result of a genetic fusion between highly homologous regions of flaA and flaB that places the active flaA promoter and the 5' coding region of flaA adjacent to previously silent regions of flaB generating a hybrid flagellin protein. Analysis of the flaA-flaB region of two such revertants reveals that a portion of this locus has undergone a rearrangement and deletion event that is unique to each revertant. Using a polymerase chain reaction (PCR) to amplify the falA-flaB locus from wild-type swimmer cells, swarmer cells and cells obtained after urinary tract infection, we uncover at least six general classes of rearrangements between flaA and flaB. Each class of rearrangement occurs within one of nine domains of homology between flaA and flaB. Rearrangement of flaA and flaB results in a hybrid flagellin protein of nearly identical size and biochemical properties, suggesting a concerted mechanism may be involved in this process. The data also reveal that the frequency and distribution of flaAB rearrangements is predicted on environmental conditions. Thus, rearrangement between flaA and flaB may be a significant virulence component of P. mirabilis in urinary tract infections.     

Cloning and sequencing of a multigene family encoding the flagellins of Methanococcus voltae.

The flagellins of Methanococcus voltae are encoded by a multigene family of four related genes (flaA, flaB1, flaB2, and flaB3). All four genes map within the same region of the genome, with the last three arranged in a direct tandem. Northern (RNA) blot and primer extension analyses of total cellular RNA indicate that all four genes are transcribed. The flaB1, flaB2, and flaB3 flagellins are transcribed as part of a large polycistronic message which encodes at least one more protein which is not a flagellin. An intercistronic stem-loop followed by a poly(T) tract located between the flaB2 and flaB3 genes appears to increase the resistance of the flaB1/flaB2 portion of this polycistronic message to digestion by endogenous RNases. The flaA gene, located approximately 600 bp upstream from the tandem, is transcribed as a separate message at very low levels. The four open reading frames encode proteins of molecular weights 23,900, 22,400, 22,800, and 25,500, much less than the Mr values of 33,000 and 31,000 for the flagellins calculated from sodium dodecyl sulfate-polyacrylamide gel electrophoresis of isolated flagellar filaments. Each flagellin contains multiple eukaryotic glycosylation signals (Arg-X-Ser/Thr), although they do not appear to be glycoproteins, and each has an 11- or 12-amino-acid leader peptide. The N termini of all four flagellins (amino acids 1 through 47 of the mature protein) are very hydrophobic, and this region shows a high degree of homology with the flagellins from Halobacterium halobium.