RESUMEN
BACKGROUND: Delayed infection, thought to be due to gradual biofilm formation, remains a feared complication after inflatable penile prosthesis (IPP) insertion. Understanding and preventing biofilm formation is necessary to prevent infections. AIM: To develop an in vitro model and compare growth of biofilm by different bacteria on IPPs and evaluate the anti-infective efficacy of the Coloplast Titan and AMS 700 InhibiZone. METHODS: Sterile IPPs (Coloplast) were cut into rings and incubated with S. epidermidis, S. aureus, P. aeruginosa, A. baumannii, or K. pneumoniae cultures in tryptic soy broth (TSB) (4 hour) to ensure adequate bacteria attachment, and then in only TSB (120 hours) to allow for biofilm formation. Rings were fixed with ethanol and biofilm measured by spectrophotometer (OD570) after crystal violet staining. This methodology was repeated for S. epidermidis and P. aeruginosa with Coloplast rings dipped in 10 ml of a 10 mg/ml Rifampin, 1 mg/ml Gentamicin, and deionized water solution and undipped AMS InhibiZone rings. Crystal violet assay (OD570) was repeated after incubation within bacteria (2 hour), and then only TSB (120 hours). OUTCOMES: The primary outcome of the study was OD570 readings, indirectly measuring biofilm mass on implant rings. RESULTS: S. epidermidis, S. aureus, A. baumannii, P. aeruginosa, and K. pneumoniae all formed significant biofilm. P. aeruginosa showed the strongest predilection to grow biofilm on IPPs. P. aeruginosa also formed significant biofilm on antibiotic-treated Coloplast and AMS rings, while S. epidermidis was inhibited. No significant difference was found in biofilm inhibition between the implants. CLINICAL TRANSLATION: Our findings suggest gram-negative bacteria may form biofilm more proficiently and quickly on IPPs than gram-positive organisms. Commonly used antibiotic treatments on IPPs may be effective against S. epidermidis but not against P. aeruginosa biofilm formation. STRENGTHS & LIMITATIONS: This is the first study comparing biofilm formation by different bacteria organisms on IPPs and the inhibitive ability of Coloplast and AMS implants against biofilm formation. Clinical data on organisms responsible for infected IPPs is needed to determine the clinical relevance of our findings. CONCLUSION: Our novel in vitro model of biofilm formation of IPPs evaluated the effect of a gentamicin/rifampin antibiotic dip on Coloplast Titan implants and the anti-infective capacity of the minocycline/rifampin precoated AMS 700 InhibiZone against S. epidermidis and P. aeruginosa. P. aeruginosa was able to grow on both antibiotic-treated implants, with no significant difference, and should continue to be a specific target of investigation to reduce delayed post-operative IPP infections. Narasimman M, Ory J, Bartra SS, et al. Evaluation of Bacteria in a Novel In Vitro Biofilm Model of Penile Prosthesis. J Sex Med 2022;19:1024-1031.
Asunto(s)
Prótesis de Pene , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Biopelículas , Gentamicinas/farmacología , Violeta de Genciana , Humanos , Rifampin/uso terapéutico , Staphylococcus aureusRESUMEN
Cellular Perforin-2 (MPEG1) is a pore-forming MACPF family protein that plays a critical role in the defense against bacterial pathogens. Macrophages, neutrophils, and several other cell types that are part of the front line of innate defenses constitutively express high levels of Perforin-2; whereas, most other cell types must be induced to express Perforin-2 by interferons (α, ß and γ) and/or PAMPs such as LPS. In this study, we demonstrate that many bacterial pathogens can limit the expression of Perforin-2 in cells normally inducible for Perforin-2 expression, while ordinarily commensal or non-pathogenic bacteria triggered high levels of Perforin-2 expression in these same cell types. The mechanisms by which pathogens suppress Perforin-2 expression was explored further using Salmonella enterica serovar Typhimurium and cultured MEFs as well as intestinal epithelial cell lines. These studies identified multiple factors required to minimize the expression of Perforin-2 in cell types inducible for Perforin-2 expression. These included the PmrAB and PhoPQ two-component systems, select LPS modification enzymes and the Type III secretion effector protein AvrA.
Asunto(s)
Lipopolisacáridos , Salmonella typhimurium , Proteínas Bacterianas/genética , Células Epiteliales , Fibroblastos , Perforina/genética , SerogrupoRESUMEN
Yersinia pestis, a Gram-negative bacterium, is the etiologic agent of plague. A hallmark of Y. pestis infection is the organism's ability to rapidly disseminate through an animal host. Y. pestis expresses the outer membrane protein, Ail (Attachment invasion locus), which is associated with host invasion and serum resistance. However, whether Ail plays a role in host dissemination remains unclear. In this study, C57BL/6J mice were challenged with a defined Y. pestis strain, KimD27, or an isogenic ail-deleted mutant derived from KimD27 via metacarpal paw pad inoculation, nasal drops, orogastric infection, or tail vein injection to mimic bubonic, pneumonic, oral, or septicemic plague, respectively. Our results showed that ail-deleted Y. pestis KimD27 lost the ability to invade host cells, leading to failed host dissemination in the pneumonic and oral plague models but not in the bubonic or septicemic plague models, which do not require invasiveness. Therefore, this study demonstrated that whether Ail plays a role in Y. pestis pathogenesis depends on the infection route.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Peste/microbiología , Factores de Virulencia/metabolismo , Virulencia , Yersinia pestis , Animales , Proteínas Bacterianas/metabolismo , Modelos Animales de Enfermedad , Pulmón/microbiología , Ratones , Ratones Endogámicos C57BL , Boca/microbiología , Yersinia pestis/metabolismo , Yersinia pestis/patogenicidadRESUMEN
Vitronectin (Vn) is a major component of blood that controls many processes central to human biology. It is a drug target and a key factor in cell and tissue engineering applications, but despite long-standing efforts, little is known about the molecular basis for its functions. Here, we define the domain organization of Vn, report the crystal structure of its carboxyl-terminal domain, and show that it harbors the binding site for the Yersinia pestis outer membrane protein Ail, which recruits Vn to the bacterial cell surface to evade human host defenses. Vn forms a single four-bladed ß/α-propeller that serves as a hub for multiple functions. The structure explains key features of native Vn and provides a blueprint for understanding and targeting this essential human protein.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Factores de Virulencia/metabolismo , Vitronectina/metabolismo , Yersinia pestis/metabolismo , Secuencia de Aminoácidos , Proteínas de la Membrana Bacteriana Externa/química , Sitios de Unión , Cristalografía por Rayos X , Humanos , Unión Proteica , Conformación Proteica , Homología de Secuencia , Factores de Virulencia/química , Vitronectina/químicaRESUMEN
Yersinia pestis, a Gram-negative bacterium and the etiologic agent of plague, has evolved from Yersinia pseudotuberculosis, a cause of a mild enteric disease. However, the molecular and biological mechanisms of how Y. pseudotuberculosis evolved to such a remarkably virulent pathogen, Y. pestis, are not clear. The ability to initiate a rapid bacterial dissemination is a characteristic hallmark of Y. pestis infection. A distinguishing characteristic between the two Yersinia species is that Y. pseudotuberculosis strains possess an O-antigen of lipopolysaccharide (LPS) while Y. pestis has lost the O-antigen during evolution and therefore exposes its core LPS. In this study, we showed that Y. pestis utilizes its core LPS to interact with SIGNR1 (CD209b), a C-type lectin receptor on antigen presenting cells (APCs), leading to bacterial dissemination to lymph nodes, spleen and liver, and the initiation of a systemic infection. We therefore propose that the loss of O-antigen represents a critical step in the evolution of Y. pseudotuberculosis into Y. pestis in terms of hijacking APCs, promoting bacterial dissemination and causing the plague.
Asunto(s)
Moléculas de Adhesión Celular/inmunología , Interacciones Huésped-Patógeno/inmunología , Lectinas Tipo C/inmunología , Lipopolisacáridos/inmunología , Peste/inmunología , Receptores de Superficie Celular/inmunología , Yersinia pestis/fisiología , Animales , Células Presentadoras de Antígenos/inmunología , Moléculas de Adhesión Celular/genética , Línea Celular , Femenino , Células HeLa , Humanos , Lectinas Tipo C/genética , Macrófagos/inmunología , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Superficie Celular/genética , Yersinia pseudotuberculosis/fisiología , Infecciones por Yersinia pseudotuberculosis/inmunologíaRESUMEN
Numerous bacterial pathogens employ specialized protein secretion machineries to directly inject anti-host proteins, termed effector proteins, into eukaryotic cells. Effector proteins carrying small phosphorylatable tags can be used to detect and quantify effector protein injection. Here, we describe the use of the ELK- and GSK-tags to detect the translocation of the Y. pestis YopE effector protein into RAW 264.7 macrophage-like cells using immunoblot analysis with phospho-specific antibodies.
Asunto(s)
Anticuerpos Fosfo-Específicos , Proteínas Bacterianas/metabolismo , Sistemas de Secreción Bacterianos/metabolismo , Epítopos , Proteínas Recombinantes de Fusión/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Fosfo-Específicos/inmunología , Proteínas Bacterianas/química , Western Blotting , Línea Celular , Epítopos/química , Epítopos/inmunología , Orden Génico , Ratones , Fosforilación , Plásmidos/genética , Transporte de Proteínas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/inmunología , Yersinia pestis/inmunología , Yersinia pestis/metabolismoRESUMEN
Yersinia pestis, the agent of plague, requires the Ail (attachment invasion locus) outer membrane protein to survive in the blood and tissues of its mammalian hosts. Ail is important for both attachment to host cells and for resistance to complement-dependent bacteriolysis. Previous studies have shown that Ail interacts with components of the extracellular matrix, including fibronectin, laminin and heparan sulfate proteoglycans, and with the complement inhibitor C4b-binding protein. Here, we demonstrate that Ail-expressing Y. pestis strains bind vitronectin - a host protein with functions in cell attachment, fibrinolysis and inhibition of the complement system. The Ail-dependent recruitment of vitronectin resulted in efficient cleavage of vitronectin by the outer membrane Pla (plasminogen activator protease). Escherichia coli DH5α expressing Y. pestis Ail bound vitronectin, but not heat-treated vitronectin. The ability of Ail to directly bind vitronectin was demonstrated by ELISA using purified refolded Ail in nanodiscs.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Interacciones Huésped-Patógeno , Factores de Virulencia/metabolismo , Vitronectina/metabolismo , Yersinia pestis/metabolismo , Proteínas Bacterianas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Humanos , Hidrólisis , Activadores Plasminogénicos/metabolismo , Unión ProteicaRESUMEN
The plague bacterium Yersinia pestis has a number of well-described strategies to protect itself from both host cells and soluble factors. In an effort to identify additional anti-host factors, we employed a transposon site hybridization (TraSH)-based approach to screen 10(5)Y. pestis mutants in an in vitro infection system. In addition to loci encoding various components of the well-characterized type III secretion system (T3SS), our screen unambiguously identified ompA as a pro-survival gene. We go on to show that an engineered Y. pestis ΔompA strain, as well as a ΔompA strain of the closely related pathogen Yersinia pseudotuberculosis, have fully functioning T3SSs but are specifically defective in surviving within macrophages. Additionally, the Y. pestis ΔompA strain was out competed by the wild-type strain in a mouse co-infection assay. Unlike in other bacterial pathogens in which OmpA can promote adherence, invasion, or serum resistance, the OmpA of Y. pestis is restricted to enhancing intracellular survival. Our data show that OmpA of the pathogenic Yersinia is a virulence factor on par with the T3SS.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Viabilidad Microbiana , Peste/microbiología , Yersinia pestis/metabolismo , Yersinia pestis/patogenicidad , Animales , Proteínas de la Membrana Bacteriana Externa/genética , Femenino , Humanos , Macrófagos/microbiología , Ratones , Ratones Endogámicos C57BL , Virulencia , Yersinia pestis/genética , Yersinia pestis/crecimiento & desarrolloRESUMEN
Yersinia pestis, a Gram-negative bacterium that causes bubonic and pneumonic plague, is able to rapidly disseminate to other parts of its mammalian hosts. Y. pestis expresses plasminogen activator (PLA) on its surface, which has been suggested to play a role in bacterial dissemination. It has been speculated that Y. pestis hijacks antigen-presenting cells, such as macrophages (MPhis) and dendritic cells, to be delivered to lymph nodes to initiate dissemination and infection. Both alveolar MPhis and pulmonary dendritic cells express a C-type lectin receptor, DEC-205 (CD205), which mediates antigen uptake and presentation. However, no ligand has been identified for DEC-205. In this study, we show that the invasion of alveolar MPhisby Y. pestis depends both in vitro and in vivo on the expression of PLA. DEC-205-expressing MPhis and transfectants, but not their negative counterparts, phagocytosed PLA-expressing Y. pestis and Escherichia coli K12 more efficiently than PLA-negative controls. The interactions between PLA-expressing bacteria and DEC-205-expressing transfectants or alveolar MPhis could be inhibited by an anti-DEC-205 antibody. Importantly, the blockage of the PLA-DEC-205 interaction reduced the dissemination of Y. pestis in mice. In conclusion, murine DEC-205 is a receptor for PLA of Y. pestis, and this host-pathogen interaction appears to play a key role in promoting bacterial dissemination.
Asunto(s)
Antígenos CD/metabolismo , Proteínas Bacterianas/metabolismo , Lectinas Tipo C/metabolismo , Activadores Plasminogénicos/metabolismo , Receptores de Superficie Celular/metabolismo , Yersinia pestis/metabolismo , Animales , Anticuerpos/inmunología , Antígenos CD/genética , Antígenos CD/inmunología , Proteínas Bacterianas/genética , Línea Celular , Cricetinae , Regulación de la Expresión Génica , Lectinas Tipo C/genética , Lectinas Tipo C/inmunología , Ligandos , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos BALB C , Antígenos de Histocompatibilidad Menor , Fagocitos/metabolismo , Plásmidos/genética , Activadores Plasminogénicos/genética , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/inmunología , Factores de Tiempo , Yersinia pestis/genéticaRESUMEN
Yersinia pestis, the causative agent of plague, must survive in blood in order to cause disease and to be transmitted from host to host by fleas. Members of the Ail/Lom family of outer membrane proteins provide protection from complement-dependent killing for a number of pathogenic bacteria. The Y. pestis KIM genome is predicted to encode four Ail/Lom family proteins. Y. pestis mutants specifically deficient in expression of each of these proteins were constructed using lambda Red-mediated recombination. The Ail outer membrane protein was essential for Y. pestis to resist complement-mediated killing at 26 and 37 degrees C. Ail was expressed at high levels at both 26 and 37 degrees C, but not at 6 degrees C. Expression of Ail in Escherichia coli provided protection from the bactericidal activity of complement. High-level expression of the three other Y. pestis Ail/Lom family proteins (the y1682, y2034, and y2446 proteins) provided no protection against complement-mediated bacterial killing. A Y. pestis ail deletion mutant was rapidly killed by sera obtained from all mammals tested except mouse serum. The role of Ail in infection of mice, Caenorhabditis elegans, and fleas was investigated.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/fisiología , Proteínas del Sistema Complemento/inmunología , Factores de Virulencia/fisiología , Yersinia pestis/inmunología , Yersinia pestis/patogenicidad , Animales , Proteínas de la Membrana Bacteriana Externa/biosíntesis , Proteínas de la Membrana Bacteriana Externa/genética , Caenorhabditis elegans/microbiología , Escherichia coli/genética , Escherichia coli/inmunología , Infecciones por Escherichia coli , Femenino , Eliminación de Gen , Expresión Génica , Ratones/microbiología , Viabilidad Microbiana , Siphonaptera/microbiología , Análisis de Supervivencia , Temperatura , Factores de Virulencia/biosíntesis , Factores de Virulencia/genética , Yersinia pestis/genéticaRESUMEN
Plague, or the Black Death, is a zoonotic disease that is spread from mammal to mammal by fleas. This mode of transmission demands that the causative agent of this disease, Yersinia pestis, is able to survive and multiply in both mammals and insects. In recent years the complete genome sequence of a number of Y. pestis strains have been determined. This sequence information indicates that Y. pestis contains a cluster of genes with homology to insecticidal toxin encoding genes of the insect pathogen Photorhabdus luminescens. Here we demonstrate that Y. pestis KIM strains produced the encoded proteins. Production of the locus-encoded proteins was dependent on a gene (yitR) encoding a member of the LysR family of transcriptional activators. Evidence suggests the proteins are type III secretion substrates. N terminal amino acids (100 to 367) of each protein fused to an epitope tag were secreted by the virulence plasmid type III secretion type. A fusion protein comprised of the N-terminus of YipB and the enzymatic active component of Bordetella pertussis adenylate cyclase (Cya) was translocated into both mammalian and insect cells. In conclusion, a new class of Y. pestis type III secreted and translocated proteins has been identified. We hypothesize that these proteins function to promote transmission of and infection by Y. pestis.
Asunto(s)
Proteínas Bacterianas/clasificación , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Yersinia pestis/genética , Yersinia pestis/patogenicidad , Adenilil Ciclasas/química , Adenilil Ciclasas/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/química , Sitios de Unión , Bordetella pertussis/enzimología , Secuencia Conservada , Epítopos , Genes Bacterianos , Genes de Insecto , Células HeLa , Humanos , Modelos Genéticos , Datos de Secuencia Molecular , Photorhabdus/genética , Photorhabdus/patogenicidad , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Homología de Secuencia de Aminoácido , Spodoptera/metabolismo , Spodoptera/microbiología , Especificidad por Sustrato , Transactivadores/genética , Yersinia pestis/clasificación , Yersinia pestis/metabolismoRESUMEN
A series of four large deletions that removed a total of ca. 36 kb of DNA from the ca. 70-kb Yersinia pestis pCD1 virulence plasmid were constructed using lambda Red-mediated recombination. Escherichia coli hha deletion mutants carrying the virulence plasmid with the deletions expressed a functional calcium-regulated type III secretion system. The E. coli hha/pCD1 system should facilitate molecular studies of the type III secretion process.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Plásmidos , Yersinia pestis/patogenicidad , Proteínas Bacterianas/genética , Calcio/metabolismo , Calcio/farmacología , Escherichia coli/metabolismo , Eliminación de Gen , Técnicas Genéticas , Plásmidos/genética , Recombinación Genética , Virulencia , Yersinia pestis/genética , Yersinia pestis/crecimiento & desarrolloRESUMEN
It is known that Yersinia pestis kills Caenorhabditis elegans by a biofilm-dependent mechanism that is similar to the mechanism used by the pathogen to block food intake in the flea vector. Using Y. pestis KIM 5, which lacks the genes that are required for biofilm formation, we show that Y. pestis can kill C. elegans by a biofilm-independent mechanism that correlates with the accumulation of the pathogen in the intestine. We used this novel Y. pestis-C. elegans pathogenesis system to show that previously known and unknown virulence-related genes are required for full virulence in C. elegans. Six Y. pestis mutants with insertions in genes that are not related to virulence before were isolated using C. elegans. One of the six mutants carried an insertion in a novel virulence gene and showed significantly reduced virulence in a mouse model of Y. pestis pathogenesis. Our results indicate that the Y. pestis-C. elegans pathogenesis system that is described here can be used to identify and study previously uncharacterized Y. pestis gene products required for virulence in mammalian systems.