scholarly journals Characterization of an operon required for growth on cellobiose in Clostridioides difficile

Microbiology ◽  
2021 ◽  
Vol 167 (8) ◽  
Author(s):  
Md Kamrul Hasan ◽  
Babita Adhikari Dhungel ◽  
Revathi Govind
Keyword(s):  
Mutant Strain ◽  
Gene Knockout ◽  
Recurrent Infection ◽  
Upstream Region ◽  
Infection Model ◽  
Clostridioides Difficile ◽  
Cellobiose Metabolism

Cellobiose metabolism is linked to the virulence properties in numerous bacterial pathogens. Here, we characterized a putative cellobiose PTS operon of Clostridiodes difficile to investigate the role of cellobiose metabolism in C. difficile pathogenesis. Our gene knockout experiments demonstrated that the putative cellobiose operon enables uptake of cellobiose into C. difficile and allows growth when cellobiose is provided as the sole carbon source in minimal medium. Additionally, using reporter gene fusion assays and DNA pulldown experiments, we show that its transcription is regulated by CelR, a novel transcriptional repressor protein, which directly binds to the upstream region of the cellobiose operon to control its expression. We have also identified cellobiose metabolism to play a significant role in C. difficile physiology as observed by the reduction of sporulation efficiency when cellobiose uptake was compromised in the mutant strain. In corroboration to in vitro study findings, our in vivo hamster challenge experiment showed a significant reduction of pathogenicity by the cellobiose mutant strain in both the primary and the recurrent infection model – substantiating the role of cellobiose metabolism in C. difficile pathogenesis.

scholarly journals Characterization of an operon required for growth on cellobiose in Clostridioides difficile

2021 ◽  
Author(s):  
Md Kamrul Hasan ◽  
Babita Adhikari Dhungel ◽  
Revathi Govind
Keyword(s):  
Mutant Strain ◽  
Gene Knockout ◽  
Recurrent Infection ◽  
In Vitro Study ◽  
Upstream Region ◽  
Infection Model ◽  
Cellobiose Metabolism

Cellobiose metabolism is linked to the virulence properties in numerous bacterial pathogens. Here, we characterized a putative cellobiose PTS operon of Clostridiodes difficile to investigate the role of cellobiose metabolism in C. difficile pathogenesis. Our gene knockout experiments demonstrated that the putative cellobiose operon enables uptake of cellobiose into C. difficile and allows growth when cellobiose is provided as the sole carbon source in minimal medium. Additionally, using reporter gene fusion assays and DNA pull-down experiments, we show that its transcription is regulated by CelR, a novel transcriptional repressor protein, which directly binds to the upstream region of the cellobiose operon to control its expression. We have also identified cellobiose metabolism to play a significant role in C. difficile physiology as observed by the reduction of sporulation efficiency when cellobiose uptake was compromised in the mutant strain. In corroboration to in vitro study findings, our in vivo hamster challenge experiment showed a significant reduction of pathogenicity by the cellobiose mutant strain in both the primary and the recurrent infection model- substantiating the role of cellobiose metabolism in C. difficile pathogenesis.

scholarly journals Reversible Daptomycin Tolerance of Adherent Staphylococci in an Implant Infection Model

2011 ◽  
Vol 55 (7) ◽  
pp. 3510-3516 ◽  
Author(s):  
Anne-K. John ◽  
Mathias Schmaler ◽  
Nina Khanna ◽  
Regine Landmann
Keyword(s):  
Treatment Failure ◽  
Mutant Strain ◽  
Calcium Ions ◽  
Biofilm Formation ◽  
Infection Model ◽  
Content Type ◽  
Wall Components

ABSTRACTDaptomycin (DAP) is bactericidal against methicillin-resistantStaphylococcus aureus(MRSA)in vitro, but it failed to eradicate MRSA in an experimental model of implant-associated infection. We therefore investigated various factors which could explain treatment failure by evaluating DAP activity, including the role of different cell wall components, adherence, biofilm, and calcium ions (Ca2+)in vitroandin vivo. In the tissue cage infection model, DAP was active only prophylactically and against low inocula. To identify the mechanisms of treatment failure, thein vitroactivity of DAP against planktonic and adherent growingS. aureusandS. epidermidismutants, differing in their capacity of biofilm formation and adherence, was determined. For planktonic staphylococci, the MIC was 0.625 μg/ml. For adherent staphylococci, DAP reduced biofilms at 30 μg/ml. However, it did not kill adherent bacteria up to 500 μg/ml, independent of biofilm biosynthesis (theicamutant strain), nuclease (thenuc1/nuc2mutant strain), LPXTG-anchored adhesin (thesrtAmutant strain), autolysin (theatlmutant strain), or alanyl-LTA (thedltAmutant strain). Resistance of adherent staphylococci was not due to mutations of adherent bacteria, since staphylococci became DAP susceptible after detachment. Phenotypic tolerance was not explained by inactivation of DAP or inability of initial Ca2+-DAP complex formation. However, the addition of up to 100 mg/liter (2.5 mmol/liter) Ca2+gradually improved bactericidal activity toward adherent staphylococciin vitroand increased the prevention rate in the cage model from 40% to 60%. In summary, adherent staphylococci are resistant to DAP killing unless Ca2+is supplemented to physiologic concentrations.

scholarly journals Activity of the Pore-Forming Virulence Factor Listeriolysin O Is Reversibly Inhibited by Naturally Occurring S-Glutathionylation

2017 ◽  
Vol 85 (4) ◽  
Author(s):  
Jonathan L. Portman ◽  
Qiongying Huang ◽  
Michelle L. Reniere ◽  
Anthony T. Iavarone ◽  
Daniel A. Portnoy
Keyword(s):  
Protein Function ◽  
Inhibitory Effect ◽  
Cysteine Residue ◽  
Infection Model ◽  
Listeriolysin O ◽  
Content Type ◽  
Pore Forming Proteins

ABSTRACT Cholesterol-dependent cytolysins (CDCs) represent a family of homologous pore-forming proteins secreted by many Gram-positive bacterial pathogens. CDCs mediate membrane binding partly through a conserved C-terminal undecapeptide, which contains a single cysteine residue. While mutational changes to other residues in the undecapeptide typically have severe effects, mutation of the cysteine residue to alanine has minor effects on overall protein function. Thus, the role of this highly conserved reactive cysteine residue remains largely unknown. We report here that the CDC listeriolysin O (LLO), secreted by the facultative intracellular pathogen Listeria monocytogenes, was posttranslationally modified by S-glutathionylation at this conserved cysteine residue and that either endogenously synthesized or exogenously added glutathione was sufficient to form this modification. When recapitulated with purified protein in vitro, this modification completely ablated the activity of LLO, and this inhibitory effect was fully reversible by treatment with reducing agents. A cysteine-to-alanine mutation in LLO rendered the protein completely resistant to inactivation by S-glutathionylation, and a mutant expressing this mutation retained full hemolytic activity. A mutant strain of L. monocytogenes expressing the cysteine-to-alanine variant of LLO was able to infect and replicate within bone marrow-derived macrophages indistinguishably from the wild type in vitro, yet it was attenuated 4- to 6-fold in a competitive murine infection model in vivo. This study suggests that S-glutathionylation may represent a mechanism by which CDC-family proteins are posttranslationally modified and regulated and help explain an evolutionary pressure to retain the highly conserved undecapeptide cysteine.

Challenges with Wound Infection Models in Drug Development

2020 ◽  
Vol 21 (13) ◽  
pp. 1301-1312 ◽  
Author(s):  
Sandeep K. Shukla ◽  
Ajay K. Sharma ◽  
Vanya Gupta ◽  
Aman Kalonia ◽  
Priyanka Shaw
Keyword(s):  
Wound Healing ◽  
Wound Infection ◽  
Chronic Wound ◽  
Wound Care ◽  
Infection Model ◽  
In Vivo Models ◽  
Infection Models

: Wound research is an evolving science trying to unfold the complex untold mechanisms behind the wound healing cascade. In particular, interest is growing regarding the role of microorganisms in both acute and chronic wound healing. Microbial burden plays an important role in the persistence of chronic wounds, ultimately resulting in delayed wound healing. It is therefore important for clinicians to understand the evolution of infection science and its various etiologies. Therefore, to understand the role of bacterial biofilm in chronic wound pathogenesis, various in vitro and in vivo models are required to investigate biofilms in wound-like settings. Infection models should be refined comprising an important signet of biofilms. These models are eminent for translational research to obtain data for designing an improved wound care formulation. However, all the existing models possess limitations and do not fit properly in the model frame for developing wound care agents. Among various impediments, one of the major drawbacks of such models is that the wound they possess does not mimic the wound a human develops. Therefore, a novel wound infection model is required which can imitate the human wounds. : This review article mainly discusses various in vitro and in vivo models showing microbial colonization, their advantages and challenges. Apart from these models, there are also present ex vivo wound infection models, but this review mainly focused on various in vitro and in vivo models available for studying wound infection in controlled conditions. This information might be useful in designing an ideal wound infection model for developing an effective wound healing formulation.

scholarly journals A Francisella tularensis SCHU S4 mutant deficient in γ-glutamyltransferase activity induces protective immunity: characterization of an attenuated vaccine candidate

Microbiology ◽  
2011 ◽  
Vol 157 (11) ◽  
pp. 3172-3179 ◽  
Author(s):  
Philip M. Ireland ◽  
Helen LeButt ◽  
Rebecca M. Thomas ◽  
Petra C. F. Oyston
Keyword(s):  
Mutant Strain ◽  
Francisella Tularensis ◽  
Protective Immunity ◽  
Attenuated Vaccine ◽  
Schu S4 ◽  
Glutamyl Transpeptidase

Francisella tularensis is an intracellular pathogen which causes tularaemia. There is no licensed vaccine currently available for prophylaxis. The γ-glutamyl transpeptidase (GGT) encoded by the ggt gene has been shown to be important for the intracellular survival of F. tularensis. In this study we have constructed a ggt deletion mutant in the highly virulent F. tularensis strain SCHU S4. Characterization of the mutant strain confirmed the function of ggt, and confirmed the role of GGT in cysteine acquisition. The mutant strain was highly attenuated both in vitro and in vivo using murine models of infection. Moreover, we have demonstrated that the attenuated mutant is able to induce protective immunity against an F. tularensis SCHU S4 challenge, and thus may be a candidate for the development of an attenuated vaccine.

scholarly journals Role of the (Mn)superoxide dismutase of Enterococcus faecalis in the in vitro interaction with microglia

Microbiology ◽  
2011 ◽  
Vol 157 (6) ◽  
pp. 1816-1822 ◽  
Author(s):  
Samuele Peppoloni ◽  
Brunella Posteraro ◽  
Bruna Colombari ◽  
Lidia Manca ◽  
Axel Hartke ◽  
...  
Keyword(s):  
Superoxide Dismutase ◽  
Enterococcus Faecalis ◽  
Stress Responses ◽  
Peritoneal Macrophages ◽  
Infection Process ◽  
Microglial Cells ◽  
Infection Model

Enterococcus faecalis is a significant human pathogen worldwide and is responsible for severe nosocomial and community-acquired infections. Although enterococcal meningitis is rare, mortality is considerable, reaching 21 %. Nevertheless, the pathogenetic mechanisms of this infection remain poorly understood, even though the ability of E. faecalis to avoid or survive phagocytic attack in vivo may be very important during the infection process. We previously showed that the manganese-cofactored superoxide dismutase (MnSOD) SodA of E. faecalis was implicated in oxidative stress responses and, interestingly, in the survival within mouse peritoneal macrophages using an in vivo–in vitro infection model. In the present study, we investigated the role of MnSOD in the interaction of E. faecalis with microglia, the brain-resident macrophages. By using an in vitro infection model, murine microglial cells were challenged in parallel with the wild-type strain JH2-2 and its isogenic sodA deletion mutant. While both strains were phagocytosed by microglia efficiently and to a similar extent, the ΔsodA mutant was found to be significantly more susceptible to microglial killing than JH2-2, as assessed by the antimicrobial protection assay. In addition, a significantly higher percentage of acidic ΔsodA-containing phagosomes was found and these also underwent enhanced maturation as determined by the expression of endolysosomal markers. In conclusion, these results show that the MnSOD of E. faecalis contributes to survival of the bacterium in microglial cells by influencing their antimicrobial activity, and this could even be important for intracellular killing in neutrophils and thus for E. faecalis pathogenesis.

scholarly journals Platelet Endothelial Cell Adhesion Molecule 1 (CD31) Is Essential for Clostridium perfringens Beta-Toxin Mediated Cytotoxicity in Human Endothelial and Monocytic Cells

Toxins ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 893
Author(s):  
Basma Tarek ◽  
Julia Bruggisser ◽  
Filippo Cattalani ◽  
Horst Posthaus
Keyword(s):  
Endothelial Cells ◽  
Clostridium Perfringens ◽  
Gene Knockout ◽  
Specific Interaction ◽  
Cell Type Specificity ◽  
Monocytic Cells ◽  
Beta Toxin

Beta toxin (CPB) is a small hemolysin beta pore-forming toxin (β-PFT) produced by Clostridium perfringens type C. It plays a central role in the pathogenesis of necro-hemorrhagic enteritis in young animals and humans via targeting intestinal endothelial cells. We recently identified the membrane protein CD31 (PECAM-1) as the receptor for CPB on mouse endothelial cells. We now assess the role of CD31 in CPB cytotoxicity against human endothelial and monocytic cells using a CRISPR/Cas9 gene knockout and an antibody blocking approach. CD31 knockout human endothelial and monocytic cells were resistant to CPB and CPB oligomers only formed in CD31-expressing cells. CD31 knockout endothelial and monocytic cells could be selectively enriched out of a polyclonal cell population by exposing them to CPB. Moreover, antibody mediated blocking of the extracellular Ig6 domain of CD31 abolished CPB cytotoxicity and oligomer formation in endothelial and monocytic cells. In conclusion, this study confirms the role of CD31 as a receptor of CPB on human endothelial and monocytic cells. Specific interaction with the CD31 molecule can thus explain the cell type specificity of CPB observed in vitro and corresponds to in vivo observations in naturally diseased animals.

scholarly journals Mycobacterium marinum Erp Is a Virulence Determinant Required for Cell Wall Integrity and Intracellular Survival

2006 ◽  
Vol 74 (6) ◽  
pp. 3125-3133 ◽  
Author(s):  
Christine L. Cosma ◽  
Kathryn Klein ◽  
Rosa Kim ◽  
Dana Beery ◽  
Lalita Ramakrishnan
Keyword(s):  
Mycobacterium Marinum ◽  
Infection Model ◽  
Leopard Frogs ◽  
Virulence Determinant ◽  
Cell Spread ◽  
Close Genetic Relative ◽  
Repetitive Protein

ABSTRACT The Mycobacterium tuberculosis exported repetitive protein (Erp) is a virulence determinant required for growth in cultured macrophages and in vivo. To better understand the role of Erp in Mycobacterium pathogenesis, we generated a mutation in the erp homologue of Mycobacterium marinum, a close genetic relative of M. tuberculosis. erp-deficient M. marinum was growth attenuated in cultured macrophage monolayers and during chronic granulomatous infection of leopard frogs, suggesting that Erp function is similarly required for the virulence of both M. tuberculosis and M. marinum. To pinpoint the step in infection at which Erp is required, we utilized a zebrafish embryo infection model that allows M. marinum infections to be visualized in real-time, comparing the erp-deficient strain to a ΔRD1 mutant whose stage of attenuation was previously characterized in zebrafish embryos. A detailed microscopic examination of infected embryos revealed that bacteria lacking Erp were compromised very early in infection, failing to grow and/or survive upon phagocytosis by host macrophages. In contrast, ΔRD1 mutant bacteria grow normally in macrophages but fail to induce host macrophage aggregation and subsequent cell-to-cell spread. Consistent with these in vivo findings, erp-deficient but not RD1-deficient bacteria exhibited permeability defects in vitro, which may be responsible for their specific failure to survive in host macrophages.

scholarly journals Skp2 Stabilizes Mcl-1 and Confers Radioresistance in Colorectal Cancer

2021 ◽  
Author(s):  
Xinfang Yu ◽  
Li Zhou ◽  
Wenbin Liu ◽  
lijun Liu ◽  
Feng Gao ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Immunohistochemical Staining ◽  
Poor Prognosis ◽  
Gene Knockout ◽  
Critical Role ◽  
Intrinsic Apoptosis ◽  
Human Colorectal Cancer

Abstract Background Overexpression of Skp2 plays a critical role in tumorigenesis and correlates with poor prognosis in human malignancies. Thus, Skp2 has been proposed as an attractive target for anti-tumor interventions. Methods The expression of Skp2 in human colorectal cancer (CRC) and the role of Skp2 in tumorigenic properties and irradiation sensitivities of CRC cells were examined by anchorage-dependent and -independent growth assays, immunoblot, flow cytometry, immunohistochemical staining, ubiquitination analysis, co-immunoprecipitation assay, CRISPR-Cas9-based gene knockout, and xenograft experiments. Results Skp2 is highly expressed in CRC patient tissues. Blocking Skp2 expression reduces the tumorigenic properties of CRC cells in vitro and in vivo. Depletion of Skp2 confers sensitivity to irradiation of CRC cells. Skp2 deficiency enhances irradiation-induced intrinsic apoptosis by facilitating E3 ligase FBW7-mediated Mcl-1 ubiquitination and degradation. Knockout of Skp2 sensitizes CRC cells to irradiation treatments in vivo. Conclusion Our findings indicate that Skp2 stabilizes Mcl-1, and targeting Skp2 in combination with traditional radiotherapy might be efficacious in treating CRC.

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