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

2012 ◽  
Vol 80 (6) ◽  
pp. 2019-2025 ◽  
Author(s):  
Ambroise Lambert ◽  
Mathieu Picardeau ◽  
David A. Haake ◽  
Rasana W. Sermswan ◽  
Amporn Srikram ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Animal Models ◽  
Acute Infection ◽  
Transposon Mutagenesis ◽  
Leptospira Interrogans ◽  
Wild Type ◽  
Hamster Model ◽  
Normal Morphology ◽  
Content Type ◽  
Periplasmic Flagella

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.

scholarly journals Leptospiral LruA Is Required for Virulence and Modulates an Interaction with Mammalian Apolipoprotein AI

2013 ◽  
Vol 81 (10) ◽  
pp. 3872-3879 ◽  
Author(s):  
Kunkun Zhang ◽  
Gerald L. Murray ◽  
Torsten Seemann ◽  
Amporn Srikram ◽  
Thanatchaporn Bartpho ◽  
...  
Keyword(s):  
Serum Protein ◽  
Serum Proteins ◽  
Acute Infection ◽  
Cellular Interactions ◽  
Wild Type ◽  
Hamster Model ◽  
Content Type ◽  
Truncation Mutant ◽  
Separation Of Proteins

ABSTRACTLeptospirosis is a worldwide zoonosis caused by spirochetes of the genusLeptospira. While understanding of pathogenesis remains limited, the development of mutagenesis inLeptospirahas provided a powerful tool for identifying novel virulence factors. LruA is a lipoprotein that has been implicated in leptospiral uveitis as a target of the immune response. In this study, twolruAmutants, M754 and M765, generated by transposon mutagenesis fromLeptospira interrogansserovar Manilae, were characterized. In M754, the transposon inserted in the middle oflruA, resulting in no detectable expression of LruA. In M765, the transposon inserted toward the 3′ end of the gene, resulting in expression of a truncated protein. LruA was demonstrated to be on the cell surface in M765 and the wild type (WT). M754, but not M765, was attenuated in a hamster model of acute infection. A search for differential binding to human serum proteins identified a serum protein of around 30 kDa bound to the wild type and the LruA deletion mutant (M754), but not to the LruA truncation mutant (M765). Two-dimensional separation of proteins from leptospiral cells incubated with guinea pig serum identified the 28-kDa apolipoprotein A-I (ApoA-I) as a major mammalian serum protein that bindsLeptospirain vitro. Interestingly, M754 (with no detectable LruA) bound more ApoA-I than did the LruA-expressing strains Manilae wild type and M765. Our data thus identify LruA as a surface-exposed leptospiral virulence factor that contributes to leptospiral pathogenesis, possibly by modulating cellular interactions with serum protein ApoA-I.

scholarly journals The Ser/Thr Kinase PrkC Participates in Cell Wall Homeostasis and Antimicrobial Resistance inClostridium difficile

2019 ◽  
Vol 87 (8) ◽  
Author(s):  
Elodie Cuenot ◽  
Transito Garcia-Garcia ◽  
Thibaut Douche ◽  
Olivier Gorgette ◽  
Pascal Courtin ◽  
...  
Keyword(s):  
Cell Wall ◽  
Type Strain ◽  
Wild Type Strain ◽  
Cell Envelope ◽  
Wild Type ◽  
Hamster Model ◽  
Content Type ◽  
Physiological Processes ◽  
Signal Transduction Networks ◽  
Mean Size

ABSTRACTClostridium difficileis the leading cause of antibiotic-associated diarrhea in adults. During infection,C. difficilemust detect the host environment and induce an appropriate survival strategy. Signal transduction networks involving serine/threonine kinases (STKs) play key roles in adaptation, as they regulate numerous physiological processes. PrkC ofC. difficileis an STK with two PASTA domains. We showed that PrkC is membrane associated and is found at the septum. We observed that deletion ofprkCaffects cell morphology with an increase in mean size, cell length heterogeneity, and presence of abnormal septa. A ΔprkCmutant was able to sporulate and germinate but was less motile and formed more biofilm than the wild-type strain. Moreover, a ΔprkCmutant was more sensitive to antimicrobial compounds that target the cell envelope, such as the secondary bile salt deoxycholate, cephalosporins, cationic antimicrobial peptides, and lysozyme. This increased susceptibility was not associated with differences in peptidoglycan or polysaccharide II composition. However, the ΔprkCmutant had less peptidoglycan and released more polysaccharide II into the supernatant. A proteomic analysis showed that the majority ofC. difficileproteins associated with the cell wall were less abundant in the ΔprkCmutant than the wild-type strain. Finally, in a hamster model of infection, the ΔprkCmutant had a colonization delay that did not significantly affect overall virulence.

scholarly journals Inactivation ofclpBin the Pathogen Leptospira interrogans Reduces Virulence and Resistance to Stress Conditions

2011 ◽  
Vol 79 (9) ◽  
pp. 3711-3717 ◽  
Author(s):  
Kristel Lourdault ◽  
Gustavo M. Cerqueira ◽  
Elsio A. Wunder ◽  
Mathieu Picardeau
Keyword(s):  
Oxidative Stress ◽  
Stress Conditions ◽  
Leptospira Interrogans ◽  
Wild Type ◽  
Content Type ◽  
General Stress Response ◽  
Growth Defects ◽  
Resistance To Stress ◽  
Increased Susceptibility

ABSTRACTLeptospira interrogansis the causative agent of leptospirosis, which is an emerging zoonotic disease. Resistance to stress conditions is largely uncharacterized for this bacterium. We therefore decided to analyze aclpBmutant that we obtained by random transposon mutagenesis. The mutant did not produce any of the two isoforms of ClpB. TheclpBmutant exhibited growth defects at 30° and 37°C and in poor nutrient medium and showed increased susceptibility to oxidative stress, whereas the genetically complemented strain was restored in ClpB expression andin vitrowild-type growth. We also showed that theclpBmutant was attenuated in virulence in an animal model of acute leptospirosis. Our findings demonstrate that ClpB is involved in the general stress response. The chaperone is also necessary, either directly or indirectly, for the virulence of the pathogenL. interrogans.

scholarly journals Oral Immunization with Escherichia coli Expressing a Lipidated Form of LigA Protects Hamsters against Challenge with Leptospira interrogans Serovar Copenhageni

2013 ◽  
Vol 82 (2) ◽  
pp. 893-902 ◽  
Author(s):  
Kristel Lourdault ◽  
Long-Chieh Wang ◽  
Ana Vieira ◽  
James Matsunaga ◽  
Rita Melo ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Oral Vaccine ◽  
Oral Immunization ◽  
Reservoir Host ◽  
Leptospira Interrogans ◽  
Renal Tubules ◽  
Hamster Model ◽  
Content Type ◽  
E Coli ◽  
Humoral Immune

ABSTRACTLeptospirosis is a potentially fatal zoonosis transmitted by reservoir host animals that harbor leptospires in their renal tubules and shed the bacteria in their urine.Leptospira interrogansserovar Copenhageni transmitted fromRattus norvegicusto humans is the most prevalent cause of urban leptospirosis. We examinedL. interrogansLigA, domains 7 to 13 (LigA7-13), as an oral vaccine delivered byEscherichia colias a lipidated, membrane-associated protein. The efficacy of the vaccine was evaluated in a susceptible hamster model in terms of the humoral immune response and survival from leptospiral challenge. Four weeks of oral administration of liveE. coliexpressing LigA7-13 improved survival from intraperitoneal (i.p.) and intradermal (i.d.) challenge byL. interrogansserovar Copenhageni strain Fiocruz L1-130 in Golden Syrian hamsters. Immunization withE. coliexpressing LigA7-13 resulted in a systemic antibody response, and a significant LigA7-13 IgG level after the first 2 weeks of immunization was completely predictive of survival 28 days after challenge. As in previous LigA vaccine studies, all immunized hamsters that survived infection had renal leptospiral colonization and histopathological changes. In summary, an oral LigA-based vaccine improved survival from leptospiral challenge by either the i.p. or i.d. route.

scholarly journals The Transcription Factor NFAT1 Participates in the Induction of CD4+ T Cell Functional Exhaustion during Plasmodium yoelii Infection

2017 ◽  
Vol 85 (9) ◽  
Author(s):  
Rachel Y. Ames ◽  
Li-Min Ting ◽  
Inessa Gendlina ◽  
Kami Kim ◽  
Fernando Macian
Keyword(s):  
T Cells ◽  
T Cell ◽  
Acute Infection ◽  
Plasmodium Yoelii ◽  
T Cell Exhaustion ◽  
Wild Type ◽  
Content Type ◽  
Cell Exhaustion ◽  
Cognate Antigen ◽  
Effector Cytokines

ABSTRACT Repeated stimulation of T cells that occurs in the context of chronic infection results in progressively reduced responsiveness of T cells to pathogen-derived antigens. This phenotype, known as T cell exhaustion, occurs during chronic infections caused by a variety of pathogens, from persistent viruses to parasites. Unlike the memory cells that typically form after successful pathogen clearance following an acute infection, exhausted T cells secrete lower levels of effector cytokines, proliferate less in response to cognate antigen, and upregulate cell surface inhibitory molecules such as PD-1 and LAG-3. The molecular events that lead to the induction of this phenotype have, however, not been fully characterized. In T cells, members of the NFAT family of transcription factors not only are responsible for the expression of many activation-induced genes but also are crucial for the induction of transcriptional programs that inhibit T cell activation and maintain tolerance. Here we show that NFAT1-deficient CD4+ T cells maintain higher proliferative capacity and expression of effector cytokines following Plasmodium yoelii infection and are therefore more resistant to P. yoelii-induced exhaustion than their wild-type counterparts. Consequently, gene expression microarray analysis of CD4+ T cells following P. yoelii-induced exhaustion shows upregulation of effector T cell-associated genes in the absence of NFAT1 compared with wild-type exhausted T cells. Furthermore, adoptive transfer of NFAT1-deficient CD4+ T cells into mice infected with P. yoelii results in increased production of antibodies to cognate antigen. Our results support the idea that NFAT1 is necessary to fully suppress effector responses during Plasmodium-induced CD4+ T cell exhaustion.

scholarly journals NonhelicalHelicobacter pyloriMutants Show Altered Gland Colonization and Elicit Less Gastric Pathology than Helical Bacteria during Chronic Infection

2019 ◽  
Vol 87 (7) ◽  
Author(s):  
Laura E. Martínez ◽  
Valerie P. O’Brien ◽  
Christina K. Leverich ◽  
Sue E. Knoblaugh ◽  
Nina R. Salama
Keyword(s):  
Chronic Infection ◽  
Cell Shape ◽  
Quantitative Imaging ◽  
Acute Infection ◽  
Three Dimensional ◽  
Wild Type ◽  
Content Type ◽  
Tissue Interactions ◽  
H Pylori ◽  
Straight Rod

ABSTRACTHalf of all humans harborHelicobacter pyloriin their stomachs. Helical cell shape is thought to facilitateH. pylori’s ability to bore into the protective mucus layer in a corkscrew-like motion, thereby enhancing colonization of the stomach.H. pyloricell shape mutants show impaired colonization of the mouse stomach, highlighting the importance of cell shape in infection. To gain a deeper understanding of how helical cell morphology promotes host colonization byH. pylori, we used three-dimensional confocal microscopy to visualize the clinical isolate PMSS1 and an isogenic straight-rod mutant (Δcsd6) within thick longitudinal mouse stomach sections. We also performed volumetric image analysis to quantify the number of bacteria residing within corpus and antral glands in addition to measuring total CFU. We found that straight rods show attenuation during acute colonization of the stomach (1 day or 1 week postinfection) as measured by total CFU. Our quantitative imaging revealed that wild-type bacteria extensively colonized antral glands at 1 week postinfection, whilecsd6mutants showed variable colonization of the antrum at this time point. During chronic infection (1 or 3 months postinfection), total CFU were highly variable but similar for wild-type and straight rods. Both wild-type and straight rods persisted and expanded in corpus glands during chronic infection. However, the straight rods showed reduced inflammation and disease progression. Thus, helical cell shape contributes to tissue interactions that promote inflammation during chronic infection, in addition to facilitating niche acquisition during acute infection.

scholarly journals Leptospira interrogans lpxDHomologue Is Required for Thermal Acclimatization and Virulence

2015 ◽  
Vol 83 (11) ◽  
pp. 4314-4321 ◽  
Author(s):  
Azad Eshghi ◽  
Jeremy Henderson ◽  
M. Stephen Trent ◽  
Mathieu Picardeau
Keyword(s):  
Outer Membrane ◽  
Type Strain ◽  
Lipid A ◽  
Wild Type Strain ◽  
Infection Model ◽  
Leptospira Interrogans ◽  
Wild Type ◽  
Content Type ◽  
Physiological Temperature ◽  
Animal Infection

ABSTRACTLeptospirosis is an emerging disease with an annual occurrence of over 1 million human cases worldwide. PathogenicLeptospirabacteria are maintained in zoonotic cycles involving a diverse array of mammals, with the capacity to survive outside the host in aquatic environments. Survival in the diverse environments encountered byLeptospiralikely requires various adaptive mechanisms. Little is known aboutLeptospiraouter membrane modification systems, which may contribute to the capacity of these bacteria to successfully inhabit and colonize diverse environments and animal hosts.Leptospirabacteria carry two genes annotated as UDP-3-O-[3-hydroxymyristoyl] glucosamineN-acyltransferase genes (la0512 and la4326 [lpxD1andlpxD2]) that in other bacteria are involved in the early steps of biosynthesis of lipid A, the membrane lipid anchor of lipopolysaccharide. Inactivation of only one of these genes, la0512/lpxD1, imparted sensitivity to the host physiological temperature (37°C) and rendered the bacteria avirulent in an animal infection model. Polymyxin B sensitivity assays revealed compromised outer membrane integrity in thelpxD1mutant at host physiological temperature, but structural analysis of lipid A in the mutant revealed only minor changes in the lipid A moiety compared to that found in the wild-type strain. In accordance with this, anin transcomplementation restored the phenotypes to a level comparable to that of the wild-type strain. These results suggest that the gene annotated aslpxD1inLeptospira interrogansplays an important role in temperature adaptation and virulence in the animal infection model.

scholarly journals In VivoEfficacy of Antimicrobials against Biofilm-Producing Pseudomonas aeruginosa

2015 ◽  
Vol 59 (8) ◽  
pp. 4974-4981 ◽  
Author(s):  
Vinay Pawar ◽  
Uliana Komor ◽  
Nadine Kasnitz ◽  
Piotr Bielecki ◽  
Marina C. Pils ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Transcriptional Profiling ◽  
Tumor Model ◽  
Experimental System ◽  
Wild Type ◽  
Content Type ◽  
Transposon Mutants ◽  
High Disease Severity

ABSTRACTPatients suffering from cystic fibrosis (CF) are commonly affected by chronicPseudomonas aeruginosabiofilm infections. This is the main cause for the high disease severity. In this study, we demonstrate thatP. aeruginosais able to efficiently colonize murine solid tumors after intravenous injection and to form biofilms in this tissue. Biofilm formation was evident by electron microscopy. Such structures could not be observed with transposon mutants, which were defective in biofilm formation. Comparative transcriptional profiling ofP. aeruginosaindicated physiological similarity of the bacteria in the murine tumor model and the CF lung. The efficacy of currently available antibiotics for treatment ofP. aeruginosa-infected CF lungs, such as ciprofloxacin, colistin, and tobramycin, could be tested in the tumor model. We found that clinically recommended doses of these antibiotics were unable to eliminate wild-typeP. aeruginosaPA14 while being effective against biofilm-defective mutants. However, colistin-tobramycin combination therapy significantly reduced the number ofP. aeruginosaPA14 cells in tumors at lower concentrations. Hence, we present a versatile experimental system that is providing a platform to test approved and newly developed antibiofilm compounds.

scholarly journals Reduced Infectivity in Cattle for an Outer Membrane Protein Mutant of Anaplasma marginale

2015 ◽  
Vol 81 (6) ◽  
pp. 2206-2214 ◽  
Author(s):  
Francy L. Crosby ◽  
Kelly A. Brayton ◽  
Forgivemore Magunda ◽  
Ulrike G. Munderloh ◽  
Karen L. Kelley ◽  
...  
Keyword(s):  
Rational Design ◽  
Anaplasma Marginale ◽  
Transposon Mutagenesis ◽  
Developmental Cycle ◽  
Wild Type ◽  
Content Type ◽  
Tick Infection ◽  
Tick Transmission

ABSTRACTAnaplasma marginaleis the causative agent of anaplasmosis in cattle. Transposon mutagenesis of this pathogen using theHimar1system resulted in the isolation of anomp10operon insertional mutant referred to as theomp10::himar1mutant. The work presented here evaluated if this mutant had morphological and/or growth rate defects compared to wild-typeA. marginale. Results showed that the morphology, developmental cycle, and growth in tick and mammalian cell cultures are similar for the mutant and the wild type. Tick transmission experiments established that tick infection levels with the mutant were similar to those with wild-typeA. marginaleand that infected ticks successfully infected cattle. However, this mutant exhibited reduced infectivity and growth in cattle. The possibility of transformingA. marginaleby transposon mutagenesis coupled within vitroandin vivoassessment of altered phenotypes can aid in the identification of genes associated with virulence. The isolation of deliberately attenuated organisms that can be evaluated in their natural biological system is an important advance for the rational design of vaccines against this species.

scholarly journals Forward Genetic Dissection of Biofilm Development byFusobacterium nucleatum: Novel Functions of Cell Division Proteins FtsX and EnvC

mBio ◽  
2018 ◽  
Vol 9 (2) ◽  
Author(s):  
Chenggang Wu ◽  
Abu Amar Mohamed Al Mamun ◽  
Truc Thanh Luong ◽  
Bo Hu ◽  
Jianhua Gu ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Division ◽  
Cell Surface ◽  
Biofilm Formation ◽  
Cell Biology ◽  
Associated Factors ◽  
Surface Dynamics ◽  
Wild Type ◽  
Content Type ◽  
Biofilm Development

ABSTRACTFusobacterium nucleatumis a key member of the human oral biofilm. It is also implicated in preterm birth and colorectal cancer. To facilitate basic studies of fusobacterial virulence, we describe here a versatile transposon mutagenesis procedure and a pilot screen for mutants defective in biofilm formation. Out of 10 independent biofilm-defective mutants isolated, the affected genes included the homologs of theEscherichia colicell division proteins FtsX and EnvC, the electron transport protein RnfA, and four proteins with unknown functions. Next, a facile new gene deletion method demonstrated that nonpolar, in-frame deletion offtsXorenvCproduces viable bacteria that are highly filamentous due to defective cell division. Transmission electron and cryo-electron microscopy revealed that the ΔftsXand ΔenvCmutant cells remain joined with apparent constriction, and scanning electron microscopy (EM) uncovered a smooth cell surface without the microfolds present in wild-type cells. FtsX and EnvC proteins interact with each other as well as a common set of interacting partners, many with unknown function. Last, biofilm development is altered when cell division is blocked by MinC overproduction; however, unlike the phenotypes of ΔftsXand ΔenvCmutants, a weakly adherent biofilm is formed, and the wild-type rugged cell surface is maintained. Therefore, FtsX and EnvC may perform novel functions inFusobacteriumcell biology. This is the first report of an unbiased approach to uncover genetic determinants of fusobacterial biofilm development. It points to an intriguing link among cytokinesis, cell surface dynamics, and biofilm formation, whose molecular underpinnings remain to be elucidated.IMPORTANCELittle is known about the virulence mechanisms and associated factors inF. nucleatum, due mainly to the lack of convenient genetic tools for this organism. We employed two efficient genetic strategies to identifyF. nucleatumbiofilm-defective mutants, revealing FtsX and EnvC among seven biofilm-associated factors. Electron microscopy established cell division defects of the ΔftsXand ΔenvCmutants, accompanied with a smooth cell surface, unlike the microfold, rugged appearance of wild-type bacteria. Proteomic studies demonstrated that FtsX and EnvC interact with each other as well as a set of common and unique interacting proteins, many with unknown functions. Importantly, blocking cell division by MinC overproduction led to formation of a weakly adherent biofilm, without alteration of the wild-type cell surface. Thus, this work links cell division and surface dynamics to biofilm development and lays a foundation for future genetic and biochemical investigations of basic cellular processes in this clinically significant pathogen.

Sign in / Sign up

Export Citation Format

Share Document