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

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.

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

Infection and Immunity ◽

10.1128/iai.00959-16 ◽

2017 ◽

Vol 85 (4) ◽

Cited By ~ 15

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.

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Challenges with Wound Infection Models in Drug Development

Current Drug Targets ◽

10.2174/1389450121666200302093312 ◽

2020 ◽

Vol 21 (13) ◽

pp. 1301-1312 ◽

Cited By ~ 1

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.

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

Microbiology ◽

10.1099/mic.0.047381-0 ◽

2011 ◽

Vol 157 (6) ◽

pp. 1816-1822 ◽

Cited By ~ 9

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.

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

Microbiology ◽

10.1099/mic.0.001079 ◽

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.

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

10.1101/2021.06.26.450058 ◽

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.

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Acetylcholine Protects against Candida albicans Infection by Inhibiting Biofilm Formation and Promoting Hemocyte Function in a Galleria mellonella Infection Model

Eukaryotic Cell ◽

10.1128/ec.00067-15 ◽

2015 ◽

Vol 14 (8) ◽

pp. 834-844 ◽

Cited By ~ 32

Author(s):

Ranjith Rajendran ◽

Elisa Borghi ◽

Monica Falleni ◽

Federica Perdoni ◽

Delfina Tosi ◽

...

Keyword(s):

Candida Albicans ◽

Biofilm Formation ◽

Galleria Mellonella ◽

Inflammatory Responses ◽

Fungal Infections ◽

Infection Model ◽

Content Type

ABSTRACT Both neuronal acetylcholine and nonneuronal acetylcholine have been demonstrated to modulate inflammatory responses. Studies investigating the role of acetylcholine in the pathogenesis of bacterial infections have revealed contradictory findings with regard to disease outcome. At present, the role of acetylcholine in the pathogenesis of fungal infections is unknown. Therefore, the aim of this study was to determine whether acetylcholine plays a role in fungal biofilm formation and the pathogenesis of Candida albicans infection. The effect of acetylcholine on C. albicans biofilm formation and metabolism in vitro was assessed using a crystal violet assay and phenotypic microarray analysis. Its effect on the outcome of a C. albicans infection, fungal burden, and biofilm formation were investigated in vivo using a Galleria mellonella infection model. In addition, its effect on modulation of host immunity to C. albicans infection was also determined in vivo using hemocyte counts, cytospin analysis, larval histology, lysozyme assays, hemolytic assays, and real-time PCR. Acetylcholine was shown to have the ability to inhibit C. albicans biofilm formation in vitro and in vivo . In addition, acetylcholine protected G. mellonella larvae from C. albicans infection mortality. The in vivo protection occurred through acetylcholine enhancing the function of hemocytes while at the same time inhibiting C. albicans biofilm formation. Furthermore, acetylcholine also inhibited inflammation-induced damage to internal organs. This is the first demonstration of a role for acetylcholine in protection against fungal infections, in addition to being the first report that this molecule can inhibit C. albicans biofilm formation. Therefore, acetylcholine has the capacity to modulate complex host-fungal interactions and plays a role in dictating the pathogenesis of fungal infections.

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Type VI secretion system and its effectors PdpC, PdpD and OpiA contribute to Francisella virulence in Galleria mellonella larvae

Infection and Immunity ◽

10.1128/iai.00579-20 ◽

2021 ◽

Author(s):

Maj Brodmann ◽

Sophie Schnider ◽

Marek Basler

Keyword(s):

Galleria Mellonella ◽

Response Regulator ◽

Infection Model ◽

Type Vi Secretion System ◽

Infectious Dose ◽

Infection Models

Francisella tularensis causes the deadly zoonotic disease tularemia in humans and is able to infect a broad range of organisms including arthropods, which are thought to play a major role in Francisella transmission. However, while mammalian in vitro and in vivo infection models are widely used to investigate Francisella pathogenicity, a detailed characterization of the major Francisella virulence factor, a non-canonical T6SS, in an arthropod in vivo infection model is missing. Here we use Galleria mellonella larvae to analyze the role of the Francisella T6SS and its corresponding effectors in F. novicida virulence. We report that G. mellonella larvae killing depends on the functional T6SS and infectious dose. In contrast to other mammalian in vivo infection models, even one of PdpC, PdpD or OpiA T6SS effectors is sufficient to kill G. mellonella larvae while sheath recycling by ClpB is dispensable. We further demonstrate that treatment by polyethylene glycol (PEG) activates Francisella T6SS in liquid culture and that this is independent of the response regulator PmrA. PEG-activated IglC secretion is dependent on T6SS structural component PdpB but independent of putative effectors PdpC, PdpD, AnmK and OpiB1-3. The results of larvae infection and secretion assay suggest that AnmK, a putative T6SS component with unknown function, interferes with OpiA-mediated toxicity but not with general T6SS activity. We establish that the easy-to-use G. mellonella larvae infection model provides new insights into function of T6SS and pathogenesis of Francisella.

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Reversible Daptomycin Tolerance of Adherent Staphylococci in an Implant Infection Model

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00172-11 ◽

2011 ◽

Vol 55 (7) ◽

pp. 3510-3516 ◽

Cited By ~ 21

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.

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Induction of Cyclooxygenase 2 by Streptococcus pyogenes Is Mediated by Cytolysins

Journal of Innate Immunity ◽

10.1159/000479153 ◽

2017 ◽

Vol 9 (6) ◽

pp. 587-597 ◽

Cited By ~ 4

Author(s):

Ulrike Blaschke ◽

Andreas Beineke ◽

Johanna Klemens ◽

Eva Medina ◽

Oliver Goldmann

Keyword(s):

Streptococcus Pyogenes ◽

Cyclooxygenase 2 ◽

Arachidonic Acid Metabolite ◽

Infection Model ◽

Synergistic Activity ◽

Wild Type ◽

Cox 2

Prostaglandin E2 (PGE2), an arachidonic acid metabolite regulating a broad range of physiological activities, is an important modulator of the severity of infection caused by Streptococcus pyogenes. Here, we investigated the role of streptococcal cytolysin S (SLS) and streptococcal cytolysin O (SLO) in the induction of cyclooxygenase-2 (COX-2), the rate-limiting enzyme in the synthesis of prostaglandins, in in vitro cultured macrophages and during in vivo infection. Macrophages were infected with S. pyogenes wild type or with the isogenic mutant strains deficient in SLS (ΔSLS), SLO (ΔSLO), or both (ΔSLS/ΔSLO), and the expression of COX-2 was determined at the transcriptional and the protein level. The results indicated that S. pyogenes induced expression of COX-2 and concomitant synthesis of PGE2 in macrophages mediated by the synergistic activity of both SLS and SLO, and involved calcium and the PKC/JNK signaling pathway. These results were validated using recombinant cytolysins. In a murine skin infection model, COX-2-positive cells were found more abundant at the site of S. pyogenes wild-type infection than at the site of infection with ΔSLS/ΔSLO mutant strain. These findings suggest that inhibitory targeting of SLS and SLO could ameliorate the adverse effects of high levels of prostaglandins during S. pyogenes infection.

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The Role of Polyphenols in the Modulation of Plasma Non-Enzymatic Antioxidant Capacity (NEAC)

International Journal for Vitamin and Nutrition Research ◽

10.1024/0300-9831/a000116 ◽

2012 ◽

Vol 82 (3) ◽

pp. 228-232 ◽

Cited By ~ 7

Author(s):

Mauro Serafini ◽

Giuseppa Morabito

Keyword(s):

Antioxidant Capacity ◽

Dietary Intervention ◽

Ex Vivo ◽

The Body ◽

Dietary Polyphenols ◽

Enzymatic Antioxidant ◽

Endogenous Antioxidant

Dietary polyphenols have been shown to scavenge free radicals, modulating cellular redox transcription factors in different in vitro and ex vivo models. Dietary intervention studies have shown that consumption of plant foods modulates plasma Non-Enzymatic Antioxidant Capacity (NEAC), a biomarker of the endogenous antioxidant network, in human subjects. However, the identification of the molecules responsible for this effect are yet to be obtained and evidences of an antioxidant in vivo action of polyphenols are conflicting. There is a clear discrepancy between polyphenols (PP) concentration in body fluids and the extent of increase of plasma NEAC. The low degree of absorption and the extensive metabolism of PP within the body have raised questions about their contribution to the endogenous antioxidant network. This work will discuss the role of polyphenols from galenic preparation, food extracts, and selected dietary sources as modulators of plasma NEAC in humans.

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