Variable Lipoprotein Hemagglutinin A Gene (vlhA) Expression in VariantMycoplasmagallisepticumStrainsIn Vivo

ABSTRACTMycoplasma gallisepticum, the primary etiologic agent of chronic respiratory disease, is a significant poultry pathogen, causing severe inflammation and leading to economic losses worldwide. Immunodominant proteins encoded by the variable lipoprotein and hemagglutinin (vlhA) gene family are thought to be important forM. gallisepticum-host interaction, pathogenesis, and immune evasion, but their exact role remains unknown. Previous work has demonstrated thatvlhAphase variation is dynamic throughout the earliest stages of infection, withvlhA3.03 being the predominantvlhAexpressed during the initial infection, and that the pattern of dominantvlhAexpression may be nonrandom and regulated by previously unrecognized mechanisms. To further investigate this gene family, we assessed thevlhAprofile of two well-characterized vaccine strains, GT5 and Mg7, avlhA3.03 mutant strain, and anM. gallisepticumpopulation expressing an alternative immunodominantvlhA. Here, we report that twoM. gallisepticumvaccine strains show differentvlhAprofiles over the first 2 days of infection compared to that of wild-type Rlow, while the population expressing an alternative immunodominantvlhAgene reverted to a profile indistinguishable from that of wild-type Rlow. Additionally, we observed a slight shift in thevlhAgene expression profile but no reduction in virulence in avlhA3.03 mutant. Taken together, these data further support the hypothesis thatM. gallisepticum vlhAgenes change in a nonstochastic temporal progression of expression and thatvlhA3.03, while preferred, is not required for virulence. Collectively, these data may be important in elucidating mechanisms of colonization and overall pathogenesis ofM. gallisepticum.

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Global Changes in Mycoplasma gallisepticum Phase-Variable Lipoprotein GenevlhAExpression duringIn VivoInfection of the Natural Chicken Host

Infection and Immunity ◽

10.1128/iai.01092-15 ◽

2015 ◽

Vol 84 (1) ◽

pp. 351-355 ◽

Cited By ~ 22

Author(s):

K. Pflaum ◽

E. R. Tulman ◽

J. Beaudet ◽

X. Liao ◽

S. J. Geary

Keyword(s):

Gene Expression ◽

Phase Variation ◽

Mycoplasma Gallisepticum ◽

Chronic Respiratory Disease ◽

Etiologic Agent ◽

Economic Losses ◽

Global Changes ◽

Phase Variable ◽

Content Type ◽

Host Interaction

Mycoplasma gallisepticumis the primary etiologic agent of chronic respiratory disease in poultry, a disease largely affecting the respiratory tract and causing significant economic losses worldwide. Immunodominant proteins encoded by members of the variable lipoprotein and hemagglutinin (vlhA) gene family are thought to be important for mechanisms ofM. gallisepticum-host interaction, pathogenesis, and immune evasion, but their exact role and the overall nature of their phase variation are unknown. To better understand these mechanisms, we assessed global transcriptomicvlhAgene expression directly fromM. gallisepticumpopulations present on tracheal mucosae during a 7-day experimental infection in the natural chicken host. Here we report differences in both dominant and minorvlhAgene expression levels throughout the first week of infection and starting as early as day 1 postinfection, consistent with a functional role not dependent on adaptive immunity for driving phase variation. Notably, data indicated that, at given time points, specificvlhAgenes were similarly dominant in multiple independent hosts, suggesting a nonstochastic temporal progression of dominantvlhAgene expression in the colonizing bacterial population. The dominant expression of a givenvlhAgene was not dependent on the presence of 12-copy GAA trinucleotide repeats in the promoter region and did not revert to the predominatevlhAgene when no longer faced with host pressures. Overall, these data indicate thatvlhAphase variation is dynamic throughout the earliest stages of infection and that the pattern of dominantvlhAexpression may be nonrandom and regulated by previously unrecognized mechanisms.

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Gene Deletion Strategy To Examine the Involvement of the Two Chondroitin Lyases in Flavobacterium columnare Virulence

Applied and Environmental Microbiology ◽

10.1128/aem.01586-15 ◽

2015 ◽

Vol 81 (21) ◽

pp. 7394-7402 ◽

Cited By ~ 15

Author(s):

Nan Li ◽

Ting Qin ◽

Xiao Lin Zhang ◽

Bei Huang ◽

Zhi Xin Liu ◽

...

Keyword(s):

Virulence Factors ◽

Gene Deletion ◽

Genetic Manipulation ◽

Bacterial Pathogen ◽

Infected Fish ◽

Economic Losses ◽

Flavobacterium Columnare ◽

Wild Type ◽

Single Strain ◽

Content Type

ABSTRACTFlavobacterium columnareis an important bacterial pathogen of freshwater fish that causes high mortality of infected fish and heavy economic losses in aquaculture. The pathogenesis of this bacterium is poorly understood, in part due to the lack of efficient methods for genetic manipulation. In this study, a gene deletion strategy was developed and used to determine the relationship between the production of chondroitin lyases and virulence. TheF. johnsoniaeompApromoter (PompA) was fused tosacBto construct a counterselectable marker forF. columnare.F. columnarecarrying PompA-sacBfailed to grow on media containing 10% sucrose. A suicide vector carrying PompA-sacBwas constructed, and a gene deletion strategy was developed. Using this approach, the chondroitin lyase-encoding genes,cslAandcslB, were deleted. The ΔcslAand ΔcslBmutants were both partially deficient in digestion of chondroitin sulfate A, whereas a double mutant (ΔcslAΔcslB) was completely deficient in chondroitin lyase activity. Cells ofF. columnarewild-type strain G4and of the chondroitin lyase-deficient ΔcslAΔcslBmutant exhibited similar levels of virulence toward grass carp in single-strain infections. Coinfections, however, revealed a competitive advantage for the wild type over the chondroitin lyase mutant. The results indicate that chondroitin lyases are not essential virulence factors ofF. columnarebut may contribute to the ability of the pathogen to compete and cause disease in natural infections. The gene deletion method developed in this study may be employed to investigate the virulence factors of this bacterium and may have wide application in many other members of the phylumBacteroidetes.

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Adhesion Protein ApfA of Actinobacillus pleuropneumoniae Is Required for Pathogenesis and Is a Potential Target for Vaccine Development

Clinical and Vaccine Immunology ◽

10.1128/cvi.00616-12 ◽

2012 ◽

Vol 20 (2) ◽

pp. 287-294 ◽

Cited By ~ 13

Author(s):

Yang Zhou ◽

Lu Li ◽

Zhaohui Chen ◽

Hong Yuan ◽

Huanchun Chen ◽

...

Keyword(s):

Vaccine Development ◽

Actinobacillus Pleuropneumoniae ◽

Etiologic Agent ◽

Host Cells ◽

Economic Losses ◽

Mouse Lung ◽

Content Type ◽

Humoral Immune ◽

Fimbrial Subunit ◽

Type Iv

ABSTRACTActinobacillus pleuropneumoniaeis the etiologic agent of porcine pleuropneumonia, which causes serious economic losses in the pig farming industry worldwide. Due to a lack of knowledge of its virulence factors and a lack of effective vaccines able to confer cross-serotype protection, it is difficult to place this disease under control. By analyzing its genome sequences, we found that type IV fimbrial subunit protein ApfA is highly conserved among different serotypes ofA. pleuropneumoniae. Our study shows that ApfA is an adhesin since its expression was greatly upregulated (135-fold) upon contact with host cells, while its deletion mutant attenuated its capability of adhesion. The inactivation ofapfAdramatically reduced the ability ofA. pleuropneumoniaeto colonize mouse lung, suggesting thatapfAis a virulence factor. Purified recombinant ApfA elicited an elevated humoral immune response and conferred robust protection against challenges withA. pleuropneumoniaeserovar 1 strain 4074 and serovar 7 strain WF83 in mice. Importantly, the anti-ApfA serum conferred significant protection against both serovar 1 and serovar 7 in mice. These studies indicate that ApfA promotes virulence through attachment to host cells, and its immunogenicity renders it a promising novel subunit vaccine candidate against infection withA. pleuropneumoniae.

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BtaE, an Adhesin That Belongs to the Trimeric Autotransporter Family, Is Required for Full Virulence and Defines a Specific Adhesive Pole of Brucella suis

Infection and Immunity ◽

10.1128/iai.01241-12 ◽

2013 ◽

Vol 81 (3) ◽

pp. 996-1007 ◽

Cited By ~ 26

Author(s):

Verónica Ruiz-Ranwez ◽

Diana M. Posadas ◽

Charles Van der Henst ◽

Silvia M. Estein ◽

Gastón M. Arocena ◽

...

Keyword(s):

Type Strain ◽

Wild Type Strain ◽

Host Cells ◽

Economic Losses ◽

Phenotypic Characterization ◽

Wild Type ◽

Content Type ◽

Bacterial Surface ◽

Cell Pole ◽

Brucella Suis

ABSTRACTBrucellais responsible for brucellosis, one of the most common zoonoses worldwide that causes important economic losses in several countries. Increasing evidence indicates that adhesion ofBrucellaspp. to host cells is an important step to establish infection. We have previously shown that the BmaC unipolar monomeric autotransporter mediates the binding ofBrucella suisto host cells through cell-associated fibronectin. Our genome analysis shows that theB. suisgenome encodes several additional potential adhesins. In this work, we characterized a predicted trimeric autotransporter that we named BtaE. By expressingbtaEin a nonadherentEscherichia colistrain and by phenotypic characterization of aB. suisΔbtaEmutant, we showed that BtaE is involved in the binding ofB. suisto hyaluronic acid. TheB. suisΔbtaEmutant exhibited a reduction in the adhesion to HeLa and A549 epithelial cells compared with the wild-type strain, and it was outcompeted by the wild-type strain in the binding to HeLa cells. The knockoutbtaEmutant showed an attenuated phenotype in the mouse model, indicating that BtaE is required for full virulence. BtaE was immunodetected on the bacterial surface at one cell pole. Using old and new pole markers, we observed that both the BmaC and BtaE adhesins are consistently associated with the new cell pole, suggesting that, inBrucella, the new pole is functionally differentiated for adhesion. This is consistent with the inherent polarization of this bacterium, and its role in the invasion process.

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The QseG Lipoprotein Impacts the Virulence of EnterohemorrhagicEscherichia coliandCitrobacter rodentiumand Regulates Flagellar Phase Variation inSalmonella entericaSerovar Typhimurium

Infection and Immunity ◽

10.1128/iai.00936-17 ◽

2018 ◽

Vol 86 (4) ◽

Cited By ~ 3

Author(s):

Elizabeth A. Cameron ◽

Charley C. Gruber ◽

Jennifer M. Ritchie ◽

Matthew K. Waldor ◽

Vanessa Sperandio

Keyword(s):

Regulatory Networks ◽

Cultured Cells ◽

Rabbit Model ◽

Phase Variation ◽

Enteric Pathogens ◽

Infection Model ◽

Phase Variable ◽

Wild Type ◽

Content Type

ABSTRACTThe QseEF histidine kinase/response regulator system modulates expression of enterohemorrhagicEscherichia coli(EHEC) andSalmonella entericaserovar Typhimurium virulence genes in response to the host neurotransmitters epinephrine and norepinephrine.qseG, which encodes an outer membrane lipoprotein, is cotranscribed withqseEFin these enteric pathogens, but there is little knowledge of its role in virulence. Here, we found that in EHEC QseG interacts with the type III secretion system (T3SS) gate protein SepL and modulates the kinetics of attaching and effacing (AE) lesion formation on tissue-cultured cells. Moreover, an EHEC ΔqseGmutant had reduced intestinal colonization in an infant rabbit model. Additionally, inCitrobacter rodentium, an AE lesion-forming pathogen like EHEC, QseG is required for full virulence in a mouse model. InS. Typhimurium, we found that QseG regulates the phase switch between the two flagellin types, FliC and FljB. In anS. Typhimurium ΔqseGmutant, the phase-variable promoter forfljBis preferentially switched into the “on” position, leading to overproduction of this phase two flagellin. In infection of tissue-cultured cells, theS. Typhimurium ΔqseGmutant provokes increased inflammatory cytokine production versus the wild type;in vivo, in a murine infection model, the ΔqseGstrain caused a more severe inflammatory response and was attenuated versus the wild-type strain. Collectively, our findings demonstrate that QseG is important for full virulence in several enteric pathogens and controls flagellar phase variation inS. Typhimurium, and they highlight both the complexity and conservation of the regulatory networks that control the virulence of enteric pathogens.

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Characterisation of a glycerol ABC transporter of Mycoplasma gallisepticum involved in pathogenicity

Applied and Environmental Microbiology ◽

10.1128/aem.03112-20 ◽

2021 ◽

Author(s):

Sara Mahdizadeh ◽

Yumiko Masukagami ◽

Chi-Wen Tseng ◽

Philip F. Markham ◽

David P. De Souza ◽

...

Keyword(s):

Abc Transporter ◽

Mycoplasma Gallisepticum ◽

Chronic Respiratory Disease ◽

Economic Losses ◽

Glycerol Metabolism ◽

Cell Phenotype ◽

Specific Gene ◽

Wild Type ◽

Stable Isotope Labelling ◽

Glycerol Uptake

MalF has been shown to be required for virulence in the important avian pathogen Mycoplasma gallisepticum. To characterise the function of MalF, predicted to be part of putative ABC transporter, we compared metabolite profiles of a mutant with a transposon inserted in malF (MalF-deficient ST mutant 04-1; ΔmalF) with those of wild type bacteria using GC/MS and LC/MS. Of those substrates likely to be transported by an ABC transport system, glycerol was detected at significantly lower abundance in the ΔmalF mutant, when compared to the wild type. Stable isotope labelling using [U-13C] glycerol and RT-qPCR analysis indicated that MalF was responsible for import of glycerol into M. gallisepticum and that, in the absence of MalF, the transcription of gtsA, which encodes a second transporter, GtsA, was upregulated, potentially to increase import of glycerol-3-phosphate into the cell to compensate for the loss of MalF. The loss of MalF appeared to have a global effect on glycerol metabolism, suggesting that it may also play a regulatory role, and cellular morphology was also affected, indicating that the change to glycerol metabolism may have a broader effect on cellular organisation. Overall, this study suggests that the reduced virulence of the ΔmalF mutant is due to perturbed glycerol uptake and metabolism, and that the operon including malF should be reannotated to golABC to reflect its function in glycerol transport. Importance Many mycoplasmas are pathogenic and cause disease in human and animals. M. gallisepticum causes chronic respiratory disease in chickens and infectious sinusitis in turkeys, resulting in economic losses in poultry industries throughout the world. To expand our knowledge about the pathogenesis of mycoplasma infections requires better understanding about the specific gene functions of these bacteria. In this study, we have characterised the metabolic function of a protein involved in pathogenicity of M. gallisepticum, as well as its effect on expression of selected genes, cell phenotype and H2O2 production. This study is a key step forward in understanding why this protein plays a key role in virulence in chickens. This study also emphasises the importance offunctional characterisation of mycoplasma proteins, using tools such as metabolomics, as prediction of function based on homology to other bacterial proteins is not always accurate.

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Mutations in fbiD (Rv2983) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01948-20 ◽

2020 ◽

Vol 65 (1) ◽

pp. e01948-20

Author(s):

Dalin Rifat ◽

Si-Yang Li ◽

Thomas Ioerger ◽

Keshav Shah ◽

Jean-Philippe Lanoix ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Clinical Evidence ◽

Clinical Samples ◽

Loss Of Function ◽

Wild Type ◽

Content Type ◽

Solid Media ◽

High Level ◽

Level Resistance

ABSTRACTThe nitroimidazole prodrugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10−5 CFU. Whole-genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, fbiC (56%), fbiA (15%), ddn (12%), fgd (4%), and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983 (fbiD), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

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The Serratia marcescens Siderophore Serratiochelin Is Necessary for Full Virulence during Bloodstream Infection

Infection and Immunity ◽

10.1128/iai.00117-20 ◽

2020 ◽

Vol 88 (8) ◽

Cited By ~ 1

Author(s):

Danelle R. Weakland ◽

Sara N. Smith ◽

Bailey Bell ◽

Ashootosh Tripathi ◽

Harry L. T. Mobley

Keyword(s):

Serratia Marcescens ◽

Bloodstream Infection ◽

Iron Acquisition ◽

Gene Clusters ◽

Clinical Isolates ◽

Wild Type ◽

Serratia Plymuthica ◽

Content Type ◽

Cas Assay ◽

Essential Nutrient

ABSTRACT Serratia marcescens is a bacterium frequently found in the environment, but over the last several decades it has evolved into a concerning clinical pathogen, causing fatal bacteremia. To establish such infections, pathogens require specific nutrients; one very limited but essential nutrient is iron. We sought to characterize the iron acquisition systems in S. marcescens isolate UMH9, which was recovered from a clinical bloodstream infection. Using RNA sequencing (RNA-seq), we identified two predicted siderophore gene clusters (cbs and sch) that were regulated by iron. Mutants were constructed to delete each iron acquisition locus individually and in conjunction, generating both single and double mutants for the putative siderophore systems. Mutants lacking the sch gene cluster lost their iron-chelating ability as quantified by the chrome azurol S (CAS) assay, whereas the cbs mutant retained wild-type activity. Mass spectrometry-based analysis identified the chelating siderophore to be serratiochelin, a siderophore previously identified in Serratia plymuthica. Serratiochelin-producing mutants also displayed a decreased growth rate under iron-limited conditions created by dipyridyl added to LB medium. Additionally, mutants lacking serratiochelin were significantly outcompeted during cochallenge with wild-type UMH9 in the kidneys and spleen after inoculation via the tail vein in a bacteremia mouse model. This result was further confirmed by an independent challenge, suggesting that serratiochelin is required for full S. marcescens pathogenesis in the bloodstream. Nine other clinical isolates have at least 90% protein identity to the UMH9 serratiochelin system; therefore, our results are broadly applicable to emerging clinical isolates of S. marcescens causing bacteremia.

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MetR-Regulated Vibrio cholerae Metabolism Is Required for Virulence

mBio ◽

10.1128/mbio.00236-12 ◽

2012 ◽

Vol 3 (5) ◽

Cited By ~ 28

Author(s):

Ryan W. Bogard ◽

Bryan W. Davies ◽

John J. Mekalanos

Keyword(s):

Amino Acid ◽

Vibrio Cholerae ◽

Virulence Factor ◽

Current Knowledge ◽

Intestinal Colonization ◽

Wild Type ◽

Pathogenic Potential ◽

Content Type ◽

Mouse Intestine

ABSTRACTLysR-type transcriptional regulators (LTTRs) are the largest, most diverse family of prokaryotic transcription factors, with regulatory roles spanning metabolism, cell growth and division, and pathogenesis. Using a sequence-defined transposon mutant library, we screened a panel ofV. choleraeEl Tor mutants to identify LTTRs required for host intestinal colonization. Surprisingly, out of 38 LTTRs, only one severely affected intestinal colonization in the suckling mouse model of cholera: the methionine metabolism regulator, MetR. Genetic analysis of genes influenced by MetR revealed thatglyA1andmetJwere also required for intestinal colonization. Chromatin immunoprecipitation of MetR and quantitative reverse transcription-PCR (qRT-PCR) confirmed interaction with and regulation ofglyA1, indicating that misregulation ofglyA1is likely responsible for the colonization defect observed in themetRmutant. TheglyA1mutant was auxotrophic for glycine but exhibited wild-type trimethoprim sensitivity, making folate deficiency an unlikely cause of its colonization defect. MetJ regulatory mutants are not auxotrophic but are likely altered in the regulation of amino acid-biosynthetic pathways, including those for methionine, glycine, and serine, and this misregulation likely explains its colonization defect. However, mutants defective in methionine, serine, and cysteine biosynthesis exhibited wild-type virulence, suggesting that these amino acids can be scavenged in vivo. Taken together, our results suggest that glycine biosynthesis may be required to alleviate an in vivo nutritional restriction in the mouse intestine; however, additional roles for glycine may exist. Irrespective of the precise nature of this requirement, this study illustrates the importance of pathogen metabolism, and the regulation thereof, as a virulence factor.IMPORTANCEVibrio choleraecontinues to be a severe cause of morbidity and mortality in developing countries. Identification ofV. choleraefactors critical to disease progression offers the potential to develop or improve upon therapeutics and prevention strategies. To increase the efficiency of virulence factor discovery, we employed a regulator-centric approach to multiplex our in vivo screening capabilities and allow whole regulons inV. choleraeto be interrogated for pathogenic potential. We identified MetR as a new virulence regulator and serine hydroxymethyltransferase GlyA1 as a new MetR-regulated virulence factor, both required byV. choleraeto colonize the infant mouse intestine. Bacterial metabolism is a prerequisite to virulence, and current knowledge of in vivo metabolism of pathogens is limited. Here, we expand the known role of amino acid metabolism and regulation in virulence and offer new insights into the in vivo metabolic requirements ofV. choleraewithin the mouse intestine.

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Assessment of Xanthomonas arboricola pv. juglandis Bacterial Load in Infected Walnut Fruits by Quantitative PCR

Plant Disease ◽

10.1094/pdis-12-18-2253-re ◽

2019 ◽

Vol 103 (10) ◽

pp. 2577-2586

Author(s):

Leonor Martins ◽

Camila Fernandes ◽

Pedro Albuquerque ◽

Fernando Tavares

Keyword(s):

Quantitative Pcr ◽

Juglans Regia ◽

Bacterial Load ◽

Etiologic Agent ◽

Economic Losses ◽

Rapid Diagnostics ◽

In Planta ◽

Chromosomal Dna ◽

Fruit Samples ◽

Xanthomonas Arboricola

Xanthomonas arboricola pv. juglandis is the etiologic agent of important walnut (Juglans regia L.) diseases, causing severe fruit drop and high economic losses in walnut production regions. Rapid diagnostics and knowledge of bacterial virulence fitness are key to hinder disease progression and apply timely phytosanitary measures. This work describes an X. arboricola pv. juglandis-specific real-time quantitative PCR (qPCR) using X. arboricola pv. juglandis-specific DNA markers to quantify the bacterial load in infected walnut plant tissues. Method validation was achieved using calibration curves obtained with serial dilutions of X. arboricola pv. juglandis chromosomal DNA and standard curves obtained from walnut samples spiked with X. arboricola pv. juglandis cells. High correlations (R2 > 0.990 and > 0.995) and low limits of detection (35 chromosomes/qPCR reaction and 2.7 CFU/qPCR reaction) were obtained for both markers considering the calibration and standard curves, respectively. Assessment of qPCR repeatability, reproducibility, and specificity allowed us to demonstrate the reliability and consistency of the method. Furthermore, in planta quantification of X. arboricola pv. juglandis bacterial load using infected walnut fruit samples showed a higher detection resolution compared with standard PCR detection. By allowing quantification of virulence fitness of distinct X. arboricola pv. juglandis strains in planta, the proposed qPCR method may contribute to assertive risk assessment of walnut diseases caused by X. arboricola pv. juglandis and ultimately help to improve phytosanitary practices.

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