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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.

Limited research has been conducted on the role of transcriptional regulators in relation to virulence in Leptospira interrogans, the etiological agent of leptospirosis. Here, we identify an L. interrogans locus that encodes a sensor protein, an anti-sigma factor antagonist, and two genes encoding proteins of unknown function. Transposon insertion into the gene encoding the sensor protein led to dampened transcription of the other 3 genes in this locus. This lb139 insertion mutant (the lb139 ؊ mutant) displayed attenuated virulence in the hamster model of infection and reduced motility in vitro. Whole-transcriptome analyses using RNA sequencing revealed the downregulation of 115 genes and the upregulation of 28 genes, with an overrepresentation of gene products functioning in motility and signal transduction and numerous gene products with unknown functions, predicted to be localized to the extracellular space. Another significant finding encompassed suppressed expression of the majority of the genes previously demonstrated to be upregulated at physiological osmolarity, including the sphingomyelinase C precursor Sph2 and LigB. We provide insight into a possible requirement for transcriptional regulation as it relates to leptospiral virulence and suggest various biological processes that are affected due to the loss of native expression of this genetic locus.

SummaryRod-shaped bacteria typically grow first via sporadic and dispersed elongation along their lateral walls and then via a combination of zonal elongation and constriction at the division site to form the poles of daughter cells. Although constriction comprises up to half of the cell cycle, its impact on cell size control and homeostasis has rarely been considered. To reveal the roles of cell elongation and constriction in bacterial size regulation during cell division, we captured the shape dynamics of Caulobacter crescentus with time-lapse structured illumination microscopy and used molecular markers as cell-cycle landmarks. We perturbed the constriction rate using a hyperconstriction mutant or fosfomycin ([(2R,3S)-3-methyloxiran-2-yl]phosphonic acid) inhibition. We report that the constriction rate contributes to both size control and homeostasis, by determining elongation during constriction and by compensating for variation in pre-constriction elongation on a single-cell basis.

Pathogenic Leptospira strains are responsible for leptospirosis, a worldwide emerging zoonotic disease. These spirochetes are unique amongst bacteria because of their corkscrew-like cell morphology and their periplasmic flagella. Motility is reported as an important virulence determinant, probably favoring entry and dissemination of pathogenic Leptospira in the host. However, proteins constituting the periplasmic flagella and their role in cell shape, motility and virulence remain poorly described. In this study, we characterized a spontaneous L. interrogans mutant strain lacking motility, correlated with the loss of the characteristic hook-shaped ends, and virulence in the animal model. Whole genome sequencing allowed the identification of one nucleotide deletion in the fliM gene resulting in a premature stop codon, thereby preventing the production of flagellar motor switch protein FliM. Genetic complementation restored cell morphology, motility and virulence comparable to those of wild type cells. Analyses of purified periplasmic flagella revealed a defect in flagella assembly, resulting in shortened flagella compared to the wild type strain. This also correlated with a lower amount of major filament proteins FlaA and FlaB. Altogether, these findings demonstrate that FliM is required for full and correct assembly of the flagella which is essential for motility and virulence.

We have developed a capillary tube assay in combination with real-time PCR to quantitate the number of chemoattracted Leptospira cells. We identified Tween 80, glucose, sucrose, and pyruvate as attractants for Leptospira cells; amino acids and vitamin B 12 were found to be nonchemotactic or weakly chemotactic. This assay has the general applicability to further our understanding of leptospiral chemotaxis. Human leptospirosis is an emerging disease with more than 1,000,000 cases occurring annually, with a case fatality rate exceeding 10% (5). Leptospira spp. belong to the spirochete phylum and consist of both saprophytic and pathogenic species, with the pathogenic species being the etiological agents of leptospirosis. Leptospira species are highly motile spirochetes, and their unique motility likely plays a role in their ability to rapidly disseminate into the host (9, 10, 16). Spirochete motility is unique as it allows these bacteria to swim in highly viscous gel-like media that slow or stop the motility of peritrichous bacteria (3,12). Leptospira motility depends on the presence of two periplasmic flagella, each arising from one of the subpolar ends of the cell without overlapping at the cell center (6). The direction of flagellar rotation is modulated by chemotaxis, which is defined by the movement of an organism toward or away from a chemical compound. The latter is called an attractant if it induces a movement toward itself and a repellant when it induces a movement away from itself. The regulation of the flagellarly based motility in relation to chemotaxis is present in many living organisms and has been well studied in model bacteria such as enterobacteria (22).Among the spirochetes, chemotaxis has been studied to a limited extent in Borrelia burgdorferi, Brachyspira hyodysenteriae, Spirochaeta aurantia, and Treponema denticola (11,15,20,24). In Leptospira spp., hemoglobin was found to be an attractant (27), but this has yet to be verified as most bacteria are attracted to small molecules, including diffusible molecules and small peptides, but not to intact proteins.In this study, we developed and modified a capillary tube assay previously used to analyze B. burgdorferi chemotaxis (2, 21) that allowed us to compare the chemotactic behavior of the saprophyte Leptospira biflexa serovar Patoc strain Patoc I with the pathogen Leptospira interrogans serovar Manilae strain L495.Development of a capillary tube assay. To carry out the assay, actively motile exponential-phase Leptospira cells in Ellinghausen-McCullough-Johnson-Harris (EMJH) culture medium (optical density at 420 nm of 0.5, which corresponds to approximately 5 ϫ 10 8 bacteria/ml) were centrifuged at low speed and gently resuspended in motility buffer consisting of 7 mM Na 2 HPO 4 , 2.2 mM KH 2 PO 4 , 17.1 mM NaCl, 4.7 mM NH 4 Cl, 14.8 M thiamine, and 0.5% bovine serum albumin (BSA). The resuspended cells were then preincubated overnight at 30°C to allow bacteria to recover motility and to deplete nutrients that were carried over by centrifugation. A...

Chemotaxis may have an important role in the infection process of pathogenic Leptospira spp.; however, little is known about the regulation of flagellar-based motility in these atypical bacteria. We generated a library of random transposon mutants of the pathogen L. interrogans, which included a mutant with insertion in the first gene of an operon containing the chemotaxis genes cheA, cheW, cheD, cheB, cheY and mcp. The disrupted gene encodes a putative histidine kinase (HK). The HK mutant was motile and virulent, but swarm plate and capillary assays suggested that chemotaxis was reduced in this mutant. Further analysis of bacterial trajectories by videomicroscopy showed that the ability of this mutant to reverse was significantly impaired in comparison to wild-type strain. Our data therefore show that this operon is required for full chemotaxis of Leptospira spp.

RÉSUMÉ. Les Monogènes récoltés chez l'Anguille Anguilla anguilla dans les eaux douces du Sud-Est de la France sont assimilés à Pseudodactylogyrus anguillae d'après les caractères morpho anatomiques de l'adulte. La chétotaxie, les cellules ciliées, le système excréteur et le hapteur de l'oncomiracidium sont décrits pour la première fois. L'ultrastructure du spermatozoïde montre un seul axonème et aucun microtubule cortical. Ces données confirment l'appartenance de ce Monogène au type Dactylogyridea. La position spécifique de P. anguillae est discutée. Une étude morphologique comparée du développement post-larvaire chez les Dactylogyridea nous amène à proposer une nouvelle famille, les Pseudodactylogyridae, pour ces Monogènes d'Anguilliformes. SUMMARY. Monogeneans were obtained from the Eel, Anguilla anguilla from fresh water in southeastern France ; on the basis of adult morphology they were identified as Pseudodactylogyrus anguillae. The chaetotaxy, ciliated cells, excretory system and haptor of the oncomiracidium are described for the first time. The ultrastructure of the spermatozoon shows a single axoneme and no cortical microtubules. These data confirm the position of this species within the Dactylogyridea. The specific status of P. anguillae is discussed. A comparative morphological study of the postlarval development of the haptor in the Dactylogyridea lead us to propose the new family Pseudo dactylogyridae for these Monogeneans of Anguilliformes.