scholarly journals Haemophilus ducreyi Seeks Alternative Carbon Sources and Adapts to Nutrient Stress and Anaerobiosis during Experimental Infection of Human Volunteers

2016 ◽  
Vol 84 (5) ◽  
pp. 1514-1525 ◽  
Author(s):  
Dharanesh Gangaiah ◽  
Xinjun Zhang ◽  
Beth Baker ◽  
Kate R. Fortney ◽  
Hongyu Gao ◽  
...  
Keyword(s):  
Transmitted Disease ◽  
Dna Recombination ◽  
Human Infection ◽  
Nutrient Stress ◽  
Haemophilus Ducreyi ◽  
Differentially Expressed ◽  
Virulence Determinants ◽  
Content Type

Haemophilus ducreyicauses the sexually transmitted disease chancroid in adults and cutaneous ulcers in children. In humans,H. ducreyiresides in an abscess and must adapt to a variety of stresses. Previous studies (D. Gangaiah, M. Labandeira-Rey, X. Zhang, K. R. Fortney, S. Ellinger, B. Zwickl, B. Baker, Y. Liu, D. M. Janowicz, B. P. Katz, C. A. Brautigam, R. S. Munson, Jr., E. J. Hansen, and S. M. Spinola, mBio 5:e01081-13, 2014,http://dx.doi.org/10.1128/mBio.01081-13) suggested thatH. ducreyiencounters growth conditions in human lesions resembling those found in stationary phase. However, howH. ducreyitranscriptionally responds to stress during human infection is unknown. Here, we determined theH. ducreyitranscriptome in biopsy specimens of human lesions and compared it to the transcriptomes of bacteria grown to mid-log, transition, and stationary phases. Multidimensional scaling showed that thein vivotranscriptome is distinct from those ofin vitrogrowth. Compared to the inoculum (mid-log-phase bacteria),H. ducreyiharvested from pustules differentially expressed ∼93 genes, of which 62 were upregulated. The upregulated genes encode homologs of proteins involved in nutrient transport, alternative carbon pathways (l-ascorbate utilization and metabolism), growth arrest response, heat shock response, DNA recombination, and anaerobiosis.H. ducreyiupregulated few genes (hgbA,flp-tad, andlspB-lspA2) encoding virulence determinants required for human infection. Most genes regulated by CpxRA, RpoE, Hfq, (p)ppGpp, and DksA, which control the expression of virulence determinants and adaptation to a variety of stresses, were not differentially expressedin vivo, suggesting that these systems are cycling on and off during infection. Taken together, these data suggest that thein vivotranscriptome is distinct from those ofin vitrogrowth and that adaptation to nutrient stress and anaerobiosis is crucial forH. ducreyisurvival in humans.

scholarly journals Mycobacterium tuberculosis Alters the Metalloprotease Activity of the COP9 Signalosome

mBio ◽  
2014 ◽  
Vol 5 (4) ◽  
Author(s):  
Lia Danelishvili ◽  
Lmar Babrak ◽  
Sasha J. Rose ◽  
Jamie Everman ◽  
Luiz E. Bermudez
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Cop9 Signalosome ◽  
Virulence Determinants ◽  
Bacterial Survival ◽  
Phagocytic Cells ◽  
Content Type ◽  
Metalloprotease Activity ◽  
Macrophage Protein

ABSTRACT Inhibition of apoptotic death of macrophages by Mycobacterium tuberculosis represents an important mechanism of virulence that results in pathogen survival both in vitro and in vivo. To identify M. tuberculosis virulence determinants involved in the modulation of apoptosis, we previously screened a transposon bank of mutants in human macrophages, and an M. tuberculosis clone with a nonfunctional Rv3354 gene was identified as incompetent to suppress apoptosis. Here, we show that the Rv3354 gene encodes a protein kinase that is secreted within mononuclear phagocytic cells and is required for M. tuberculosis virulence. The Rv3354 effector targets the metalloprotease (JAMM) domain within subunit 5 of the COP9 signalosome (CSN5), resulting in suppression of apoptosis and in the destabilization of CSN function and regulatory cullin-RING ubiquitin E3 enzymatic activity. Our observation suggests that alteration of the metalloprotease activity of CSN by Rv3354 possibly prevents the ubiquitin-dependent proteolysis of M. tuberculosis-secreted proteins. IMPORTANCE Macrophage protein degradation is regulated by a protein complex called a signalosome. One of the signalosomes associated with activation of ubiquitin and protein labeling for degradation was found to interact with a secreted protein from M. tuberculosis, which binds to the complex and inactivates it. The interference with the ability to inactivate bacterial proteins secreted in the phagocyte cytosol may have crucial importance for bacterial survival within the phagocyte.

scholarly journals Haemophilus ducreyi-Induced Interleukin-10 Promotes a Mixed M1 and M2 Activation Program in Human Macrophages

2012 ◽  
Vol 80 (12) ◽  
pp. 4426-4434 ◽  
Author(s):  
Wei Li ◽  
Barry P. Katz ◽  
Stanley M. Spinola
Keyword(s):  
Macrophage Polarization ◽  
Interleukin 10 ◽  
Human Infection ◽  
Haemophilus Ducreyi ◽  
Mitogen Activated Protein Kinase ◽  
Surface Markers ◽  
Microbial Infection ◽  
Bacterial Killing ◽  
Content Type

ABSTRACTDuring microbial infection, macrophages are polarized to classically activated (M1) or alternatively activated (M2) cells in response to microbial components and host immune mediators. Proper polarization of macrophages is critical for bacterial clearance. To study the role of macrophage polarization duringHaemophilus ducreyiinfection, we analyzed a panel of macrophage surface markers in skin biopsy specimens of pustules obtained from experimentally infected volunteers. Lesional macrophages expressed markers characteristic of both M1 and M2 polarization. Monocyte-derived macrophages (MDM) also expressed a mixed M1 and M2 profile of surface markers and cytokines/chemokines upon infection withH. ducreyiin vitro. Endogenous interleukin 10 (IL-10) produced by infected MDM downregulated and enhanced expression of several M1 and M2 markers, respectively. Bacterial uptake, mediated mainly by class A scavenger receptors, and activation of mitogen-activated protein kinase and phosphoinositide 3-kinase signaling pathways were required forH. ducreyi-induced IL-10 production in MDM. Compared to M1 cells, IL-10-polarized M2 cells displayed enhanced phagocytic activity againstH. ducreyiand similar bacterial killing. Thus, IL-10-modulated macrophage polarization may contribute toH. ducreyiclearance during human infection.

scholarly journals Adaptive Strategies and Pathogenesis of Clostridium difficile fromIn VivoTranscriptomics

2013 ◽  
Vol 81 (10) ◽  
pp. 3757-3769 ◽  
Author(s):  
Claire Janoir ◽  
Cécile Denève ◽  
Sylvie Bouttier ◽  
Frédéric Barbut ◽  
Sandra Hoys ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Stress Responses ◽  
Differentially Expressed ◽  
Content Type ◽  
Host Interactions ◽  
Colonization Factor ◽  
Genes Encoding ◽  
Colonization Process

ABSTRACTClostridium difficileis currently the major cause of nosocomial intestinal diseases associated with antibiotic therapy in adults. In order to improve our knowledge ofC. difficile-host interactions, we analyzed the genome-wide temporal expression ofC. difficile630 genes during the first 38 h of mouse colonization to identify genes whose expression is modulatedin vivo, suggesting that they may play a role in facilitating the colonization process. In the ceca of theC. difficile-monoassociated mice, 549 genes of theC. difficilegenome were differentially expressed compared to their expression duringin vitrogrowth, and they were distributed in several functional categories. Overall, our results emphasize the roles of genes involved in host adaptation. Colonization results in a metabolic shift, with genes responsible for the fermentation as well as several other metabolic pathways being regulated inversely to those involved in carbon metabolism. In addition, several genes involved in stress responses, such as ferrous iron uptake or the response to oxidative stress, were regulatedin vivo. Interestingly, many genes encoding conserved hypothetical proteins (CHP) were highly and specifically upregulatedin vivo. Moreover, genes for all stages of sporulation were quickly inducedin vivo, highlighting the observation that sporulation is central to the persistence ofC. difficilein the gut and to its ability to spread in the environment. Finally, we inactivated two genes that were differentially expressedin vivoand evaluated the relative colonization fitness of the wild-type and mutant strains in coinfection experiments. We identified a CHP as a putative colonization factor, supporting the suggestion that thein vivotranscriptomic approach can unravel newC. difficilevirulence genes.

scholarly journals Selective Activation of sar Promoters with the Use of Green Fluorescent Protein Transcriptional Fusions as the Detection System in the Rabbit Endocarditis Model

1998 ◽  
Vol 66 (12) ◽  
pp. 5988-5993 ◽  
Author(s):  
A. L. Cheung ◽  
Cynthia C. Nast ◽  
A. S. Bayer
Keyword(s):  
Fluorescence Microscopy ◽  
Fluorescent Protein ◽  
Detection System ◽  
Immunoblot Analysis ◽  
Growth Cycle ◽  
Differentially Expressed ◽  
Virulence Determinants ◽  
The Individual

ABSTRACT The global regulatory locus sar is composed of three overlapping transcripts initiated from a triple-promoter system (designated P1, P3, and P2). To explore if the individualsar promoters are differentially expressed in vitro and in vivo, we constructed a shuttle plasmid (pALC1434) containing a promoterless gfp UV gene (a gfpderivative [Clontech]) preceded by a polylinker region. Recombinant shuttle vectors containing individual sar promoters upstream of the gfp UV reporter gene were then introduced into Staphylococcus aureus RN6390. Northern and immunoblot analysis revealed that P1 is stronger than the P2 and P3 promoters in vitro. Additionally, the levels of thegfp UV transcript driven by individualsar promoters also correlated with the growth cycle dependency of these promoters in liquid cultures, thus suggesting the utility of pALC1434 as a vehicle for reporter fusion. Using the rabbit endocarditis model, we examined the expression of these three GFPUV fusions in vivo by fluorescence microscopy of infected cardiac vegetations 24 h after initial intravenous challenge. Similar to the in vitro findings, P1 was activated both in the center and on the surface of the vegetations. In contrast, the P3 promoter was silent both in vivo and in vitro as determined by fluorescence microscopy. Remarkably, P2 was silent in vitro but became highly activated in vivo. In particular, the sar P2 promoter was activated on the surface of the vegetation but not in the center of the lesion. These data imply that in vivo promoter activation of sar differed from that observed in vitro. Moreover, the individual sar promoters may be differentially expressed in different areas within the same anatomic niche, presumably reflecting the microbial physiological response to distinct host microenvironments. As the sar locus controls the synthesis of both extracellular and cell wall virulence determinants, these promoter-gfp UV constructs should be useful to characterize many aspects of S. aureus gene regulation in vivo.

scholarly journals The Mouse Inhalation Model ofCryptococcus neoformansInfection Recapitulates Strain Virulence in Humans and Shows that Closely Related Strains Can Possess Differential Virulence

2019 ◽  
Vol 87 (5) ◽  
Author(s):  
Liliane Mukaremera ◽  
Tami R. McDonald ◽  
Judith N. Nielsen ◽  
Christopher J. Molenaar ◽  
Andrew Akampurira ◽  
...  
Keyword(s):  
Survival Time ◽  
Cryptococcal Meningitis ◽  
Strong Association ◽  
Human Infection ◽  
Sequence Type ◽  
Disease Outcome ◽  
Content Type ◽  
Fungal Burden

ABSTRACTCryptococcal meningitis (CM) causes high rates of HIV-related mortality, yet theCryptococcusfactors influencing patient outcome are not well understood. Pathogen-specific traits, such as the strain genotype and degree of antigen shedding, are associated with the clinical outcome, but the underlying biology remains elusive. In this study, we examined factors determining disease outcome in HIV-infected cryptococcal meningitis patients infected withCryptococcus neoformansstrains with the same multilocus sequence type (MLST). Both patient mortality and survival were observed during infections with the same sequence type. Disease outcome was not associated with the patient CD4 count. Patient mortality was associated with higher cryptococcal antigen levels, the cerebrospinal fluid (CSF) fungal burden by quantitative culture, and low CSF fungal clearance. The virulence of a subset of clinical strains with the same sequence type was analyzed using a mouse inhalation model of cryptococcosis. We showed a strong association between human and mouse mortality rates, demonstrating that the mouse inhalation model recapitulates human infection. Similar to human infection, the ability to multiplyin vivo, demonstrated by a high fungal burden in lung and brain tissues, was associated with mouse mortality. Mouse survival time was not associated with singleC. neoformansvirulence factorsin vitroorin vivo; rather, a trend in survival time correlated with a suite of traits. These observations show that MLST-derived genotype similarities betweenC. neoformansstrains do not necessarily translate into similar virulence either in the mouse model or in human patients. In addition, our results show thatin vitroassays do not fully reproducein vivoconditions that influenceC. neoformansvirulence.

scholarly journals Bypassing Phase Variation of Lipooligosaccharide (LOS): Using Heptose 1 Glycan Mutants To Establish Widespread Efficacy of Gonococcal Anti-LOS Monoclonal Antibody 2C7

2019 ◽  
Vol 88 (2) ◽  
Author(s):  
Srinjoy Chakraborti ◽  
Sunita Gulati ◽  
Bo Zheng ◽  
Frank J. Beurskens ◽  
Janine Schuurman ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Polymorphonuclear Leukocytes ◽  
Phase Variation ◽  
Human Infection ◽  
Phase Variable ◽  
Content Type ◽  
Human Igg1 ◽  
And Function

ABSTRACT The sialylatable lacto-N-neotetraose (LNnT; Gal-GlcNAc-Gal-Glc) moiety from heptose I (HepI) of the lipooligosaccharide (LOS) of Neisseria gonorrhoeae undergoes positive selection during human infection. Lactose (Gal-Glc) from HepII, although phase variable, is commonly expressed in humans; loss of HepII lactose compromises gonococcal fitness in mice. Anti-LOS monoclonal antibody (MAb) 2C7, a promising antigonococcal immunotherapeutic that elicits complement-dependent bactericidal activity and attenuates gonococcal colonization in mice, recognizes an epitope comprised of lactoses expressed simultaneously from HepI and HepII. Glycan extensions beyond lactose on HepI modulate binding and function of MAb 2C7 in vitro. Here, four gonococcal LOS mutants, each with lactose from HepII but fixed (unable to phase-vary) LOS HepI glycans extended beyond the lactose substitution of HepI (lactose alone, Gal-lactose, LNnT, or GalNAc-LNnT), were used to define how HepI glycan extensions affect (i) mouse vaginal colonization and (ii) efficacy in vitro and in vivo of a human IgG1 chimeric derivative of MAb 2C7 (2C7-Ximab) with a complement-enhancing E-to-G Fc mutation at position 430 (2C7-Ximab-E430G). About 10-fold lower 2C7-Ximab-E430G concentrations achieved similar complement-dependent killing of three gonococcal mutants with glycan extensions beyond lactose-substituted HepI (lactose alone, LNnT, or GalNAc-LNnT) as 2C7-Ximab (unmodified Fc). The fourth mutant (Gal-lactose) resisted direct complement-dependent killing but was killed approximately 70% by 2C7-Ximab-E430G in the presence of polymorphonuclear leukocytes and complement. Only mutants with (sialylatable) LNnT from HepI colonized mice for >3 days, reiterating the importance of LNnT sialylation for infection. 2C7-Ximab-E430G significantly attenuated colonization caused by the virulent mutants.

scholarly journals Evaluation of CpxRA as a Therapeutic Target for UropathogenicEscherichia coliInfections

2018 ◽  
Vol 86 (3) ◽  
pp. e00798-17 ◽  
Author(s):  
Lana Dbeibo ◽  
Julia J. van Rensburg ◽  
Sara N. Smith ◽  
Kate R. Fortney ◽  
Dharanesh Gangaiah ◽  
...  
Keyword(s):  
Phosphatase Activity ◽  
Virulence Determinants ◽  
Membrane Repair ◽  
Single Strain ◽  
Content Type ◽  
Phosphatase Inhibitors ◽  
Genes Encoding ◽  
Tract Infections

ABSTRACTCpxRA is an envelope stress response system found in all members of the familyEnterobacteriaceae; CpxA has kinase activity for CpxR and phosphatase activity for phospho-CpxR, a transcription factor. CpxR also accepts phosphate groups from acetyl phosphate, a glucose metabolite. Activation of CpxR increases the transcription of genes encoding membrane repair and downregulates virulence determinants. We hypothesized that activation of CpxR could serve as an antimicrobial/antivirulence strategy and discovered compounds that activate CpxR inEscherichia coliby inhibiting CpxA phosphatase activity. As a prelude to testing such compoundsin vivo, here we constructedcpxA(in the presence of glucose, CpxR is activated because of a lack of CpxA phosphatase) andcpxR(system absent) deletion mutants of uropathogenicE. coli(UPEC) CFT073. By RNA sequencing, few transcriptional differences were noted between thecpxRmutant and its parent, but in thecpxAmutant, several UPEC virulence determinants were downregulated, including thefimandpapoperons, and it exhibited reduced mannose-sensitive hemagglutination of guinea pig red blood cellsin vitro. In competition experiments with mice, both mutants were less fit than the parent in the urine, bladder, and kidney; these fitness defects were complemented intrans. Unexpectedly, in single-strain challenges, only thecpxAmutant was attenuated for virulence in the kidney but not in the bladder or urine. For thecpxAmutant, this may be due to the preferential use of amino acids over glucose as a carbon source in the bladder and urine by UPEC. These studies suggest that CpxA phosphatase inhibitors may have some utility for treating complex urinary tract infections.

scholarly journals Genome Integration and Excision by a New Streptomyces Bacteriophage, ϕJoe

2016 ◽  
Vol 83 (5) ◽  
Author(s):  
Paul C. M. Fogg ◽  
Joshua A. Haley ◽  
W. Marshall Stark ◽  
Margaret C. M. Smith
Keyword(s):  
Synthetic Biology ◽  
High Efficiency ◽  
Dna Recombination ◽  
Streptomyces Venezuelae ◽  
Site Specific ◽  
Content Type ◽  
Wide Range ◽  
Serine Integrase

ABSTRACT Bacteriophages are the source of many valuable tools for molecular biology and genetic manipulation. In Streptomyces, most DNA cloning vectors are based on serine integrase site-specific DNA recombination systems derived from phage. Because of their efficiency and simplicity, serine integrases are also used for diverse synthetic biology applications. Here, we present the genome of a new Streptomyces phage, ϕJoe, and investigate the conditions for integration and excision of the ϕJoe genome. ϕJoe belongs to the largest Streptomyces phage cluster (R4-like) and encodes a serine integrase. The attB site from Streptomyces venezuelae was used efficiently by an integrating plasmid, pCMF92, constructed using the ϕJoe int-attP locus. The attB site for ϕJoe integrase was occupied in several Streptomyces genomes, including that of S. coelicolor, by a mobile element that varies in gene content and size between host species. Serine integrases require a phage-encoded recombination directionality factor (RDF) to activate the excision reaction. The ϕJoe RDF was identified, and its function was confirmed in vivo. Both the integrase and RDF were active in in vitro recombination assays. The ϕJoe site-specific recombination system is likely to be an important addition to the synthetic biology and genome engineering toolbox. IMPORTANCE Streptomyces spp. are prolific producers of secondary metabolites, including many clinically useful antibiotics. Bacteriophage-derived integrases are important tools for genetic engineering, as they enable integration of heterologous DNA into the Streptomyces chromosome with ease and high efficiency. Recently, researchers have been applying phage integrases for a variety of applications in synthetic biology, including rapid assembly of novel combinations of genes, biosensors, and biocomputing. An important requirement for optimal experimental design and predictability when using integrases, however, is the need for multiple enzymes with different specificities for their integration sites. In order to provide a broad platform of integrases, we identified and validated the integrase from a newly isolated Streptomyces phage, ϕJoe. ϕJoe integrase is active in vitro and in vivo. The specific recognition site for integration is present in a wide range of different actinobacteria, including Streptomyces venezuelae, an emerging model bacterium in Streptomyces research.

scholarly journals 2887. Identifying Candida albicans Transcription Factors (TFs) That Regulate Pathogenesis of Intra-abdominal Candidiasis (IAC) by Screening a Deletion Mutant Library

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S77-S77
Author(s):  
Cornelius J Clancy ◽  
Shaoji Cheng ◽  
Kevin Squires ◽  
Minh-Hong Nguyen
Keyword(s):  
Response Regulator ◽  
Transcriptional Profiling ◽  
Copper Metabolism ◽  
Homozygous Deletion ◽  
Human Infection ◽  
Virulence Determinants ◽  
Mutant Library ◽  
Ph Response

Abstract Background IAC is a common manifestation of invasive candidiasis, but its pathogenesis is poorly understood. We developed a mouse model of C. albicans IAC, in which disease progresses from peritonitis to abscesses (IAA) in a manner that recapitulates human infection. Our goal was to use the model to identify C. albicans TFs that regulate virulence during IAC. Methods We screened a signature-tagged library (48 unique oligonucleotide markers) of homozygous deletion mutants for 165 C. albicans TF genes, created in duplicate in strain SC5314 (S. Noble). Mice were infected intra-peritoneally in triplicate with pools of 24 mutants and wild-type, and strains harvested at 72 hours in IAA. Results Twenty-one TF mutants were significantly attenuated for virulence in both libraries, and 2 TF mutants were significantly more virulent in both libraries, as measured by tissue burdens (figure). Biologic processes over-represented among attenuated mutants were regulation of pH responses, biofilm, hyphal formation, echinocandin responses, and copper metabolism. pH responses are likely to be crucial to pathogenesis of IAC, as C. albicans transitions from pH 8 during peritonitis to pH 6.8 within IAA. 9 pH response regulators contributing to virulence included RIM101, STP2 (alkaline), ASH1, SFL1, SFL2 (neutral), MNL1, SKO1, PHO4 (weak acid), and CSR1 (acid). We created rim101 null mutant and reconstitution strains, and demonstrated that the gene was essential for complete virulence during peritonitis and IAA. Transcriptional profiling of strains by RT-PCR during peritonitis and in vitro showed both conserved and rewired Rim101 targets. SAP5, which encodes an aspartyl protease, is a major Rim101 target in vivo and in vitro; over-expression of SAP5 in rim101 restored virulence during IAA at 3, 7, and 10 days, but not during peritonitis. Other pH regulatory TF genes are currently being validated as virulence determinants, and pathway relationships between MNL1, SKO1, and PHO4 during IAA formation are being explored through epistasis approaches. Conclusion Screening of a C. albicans TF mutant library identified pH responses and other biologic processes as important during pathogenesis of IAC. Rim101, an alkaline pH response regulator, contributes to both peritonitis and IAA, the latter at least in part through its effects on Sap5. Disclosures All Authors: No reported Disclosures.

scholarly journals Chancroid and Haemophilus ducreyi: an update.

1995 ◽  
Vol 8 (3) ◽  
pp. 357-375 ◽  
Author(s):  
D L Trees ◽  
S A Morse
Keyword(s):  
The United States ◽  
Selective Medium ◽  
Molecular Techniques ◽  
Haemophilus Ducreyi ◽  
Virulence Determinants ◽  
Transmitted Infection ◽  
In Vivo Models ◽  
Clinical Appearance

Haemophilus ducreyi is a fastidious gram-negative bacillus that causes the sexually transmitted infection chancroid. Chancroid is a major genital ulcerative disease in Africa, Southeast Asia, the Caribbean, and Latin America and is of increasing concern in the United States. Genital ulcerative disease and chancroid in particular have been associated with facilitating the transmission of human immunodeficiency virus. The diagnosis of chancroid based on the clinical appearance of the genital lesion or on the isolation of H. ducreyi on selective medium is relatively insensitive. However, recent advances in nonculture diagnostic tests have enhanced our ability to diagnose chancroid. There has been renewed interest in understanding the pathogenesis of H. ducreyi. In vitro and in vivo models have been developed to help identify important virulence determinants. Through the use of biochemical and molecular techniques, macromolecular components that may be important in virulence have been identified.

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