Biomedical subjects

Joan Mecsas

Publications and source records attributed to Joan Mecsas.

10 recordsLinked to original sources

PubMed2006

Intranasal inoculation of mice with Yersinia pseudotuberculosis causes a lethal lung infection that is dependent on Yersinia outer proteins and PhoP.

Yersinia pseudotuberculosis infects many mammals and birds including humans, livestock, and wild rodents and can be recovered from the lungs of infected animals. To determine the Y. pseudotuberculosis factors important for growth during lung infection, we developed an intranasal model of infection in mice. Following intranasal inoculation, we monitored both bacterial growth in lungs and dissemination to systemic tissues. Intranasal inoculation with as few as 18 CFU of Y. pseudotuberculosis caused a lethal lung infection in some mice. Over the course of 7 days, wild-type Y. pseudotuberculosis replicated to nearly 1 x 10(8) CFU/g of lung in BALB/c mice, induced histopathology in lungs consistent with pneumonia, but disseminated sporadically to other tissues. In contrast, a Delta yopB deletion strain was attenuated in this model, indicating that translocation of Yersinia outer proteins (Yops) is essential for virulence. Additionally, a Delta yopH null mutant failed to grow to wild-type levels by 4 days postintranasal inoculation, but deletions of any other single effector YOP did not attenuate lung colonization 4 days postinfection. Strains with deletions in yopH and any one of the other known effector yop genes were more attenuated that the Delta yopH strain, indicating a unique role for yopH in lungs. In summary, we have characterized the progression of a lung infection with an enteric Yersinia pathogen and shown that YopB and YopH are important in lung colonization and dissemination. Furthermore, this lung infection model with Y. pseudotuberculosis can be used to test potential therapeutics against Yersinia and other gram-negative infections in lungs.

Administration, Intranasal↗

PubMed2006

Mutations in the Yersinia pseudotuberculosis type III secretion system needle protein, YscF, that specifically abrogate effector translocation into host cells.

The trafficking of effectors, termed Yops, from Yersinia spp. into host cells is a multistep process that requires the type III secretion system (TTSS). The TTSS has three main structural parts: a base, a needle, and a translocon, which work together to ensure the polarized movement of Yops directly from the bacterial cytosol into the host cell cytosol. To understand the interactions that take place at the interface between the tip of the TTSS needle and the translocon, we developed a screen to identify mutations in the needle protein YscF that separated its function in secretion from its role in translocation. We identified 25 translocation-defective (TD) yscF mutants, which fall into five phenotypic classes. Some classes exhibit aberrant needle structure and/or reduced levels of Yop secretion, consistent with known functions for YscF. Strikingly, two yscF TD classes formed needles and secreted Yops normally but displayed distinct translocation defects. Class I yscF TD mutants showed diminished pore formation, suggesting incomplete pore insertion and/or assembly. Class II yscF TD mutants formed pores but showed nonpolar translocation, suggesting unstable needle-translocon interactions. These results indicate that YscF functions in Yop secretion and translocation can be genetically separated. Furthermore, the identification of YscF residues that are required for the assembly of the translocon and/or productive interactions with the translocon has allowed us to initiate the mapping of the needle-translocon interface.

Bacterial Proteins↗

PubMed2006

Oral inoculation with Type III secretion mutants of Yersinia pseudotuberculosis provides protection from oral, intraperitoneal, or intranasal challenge with virulent Yersinia.

The enteric pathogen Yersinia pseudotuberculosis (Yptb) causes gastroenteritis, mesenteric lymphadenitis, and systemic infections in humans, livestock, and wild animals. Yptb Type III secretion system (pTTSS) mutants efficiently colonize lymphoid tissues, but not the gastrointestinal tract, spleen, or liver. Here, we show that a single oral inoculation of pTTSS mutants prevents morbidity in almost 100% of mice challenged intragastrically with virulent Yptb. In addition, a single oral inoculation of a pTTSS mutant protected 50% of mice challenged intraperitoneally or intranasally with virulent Yptb. In addition, the intranasally challenged mice that succumbed to infection lived significantly longer than non-immunized mice. Thus, pTTSS mutants can function as live attenuated vaccine when delivered orally. Potential uses for these attenuated strains include use as a livestock vaccine, a rodent plague control reagent in endemic areas around the world, and a vector for delivery of other antigens to the mesenteric lymph nodes.

Administration, Oral↗

PubMed2006

A non-invasive quantitative assay to measure murine intestinal inflammation using the neutrophil marker lactoferrin.

Intestinal inflammation in mice is most frequently assessed by histology or FACS, processes that necessitate sacrificing mice. We developed a lactoferrin ELISA for murine feces to quantify intestinal inflammation in mice with enteric infections or colitis. Levels of fecal lactoferrin, a protein secreted by activated neutrophils, were consistent with neutrophil infiltration as assessed by histology, indicating that this fecal lactoferrin ELISA is a good alternative to histology. The fecal lactoferrin ELISA provides a non-invasive, quantitative assessment of intestinal inflammation, which should facilitate longitudinal studies of the development of and/or therapies reducing intestinal inflammation in individual mice and reduce the number of mice needed for such studies.

Animals↗

PubMed2006

Yersinia pseudotuberculosis disseminates directly from a replicating bacterial pool in the intestine.

Dissemination of Yersinia pseudotuberculosis within mice after oral inoculation was analyzed. Y. pseudotuberculosis translocated to organs such as the liver and spleen shortly after oral inoculation, but was quickly cleared. In contrast, a second temporally distinct bacterial translocation event resulted in successful hepatosplenic replication of the bacteria. Replicating pools of bacteria could be established in these organs in mouse mutants that lacked Peyer's patches. These animals frequently had sterile mesenteric lymph nodes, a finding consistent with translocation taking place independently of regional lymph node colonization. In further contradiction to accepted models for dissemination of enteropathogens, clonal analysis revealed that bacteria causing disease in the spleen and liver of C57BL/6J mice were derived from populations located outside the intestinal lymph nodes. Replication of bacteria in the intestine before translocation appeared critical for dissemination, as transient selective suppression by streptomycin of bacterial growth in the intestine delayed dissemination of Y. pseudotuberculosis. These results collectively indicate that hepatosplenic colonization appears intimately connected with the ability of Y. pseudotuberculosis to successfully establish replication in the intestinal lumen and does not result from ordered spread leading from the intestine to regional lymph nodes before dissemination.

Animals↗

PubMed2006

The proinflammatory response induced by wild-type Yersinia pseudotuberculosis infection inhibits survival of yop mutants in the gastrointestinal tract and Peyer's patches.

Single-strain infections and coinfections are frequently used to assess roles of virulence factors in infected tissues. After oral inoculation of mice, Yersinia pseudotuberculosis yopE and yopH mutants colonize the intestines and Peyer's patches in single-strain infections but fail to persist in competition with wild-type Y. pseudotuberculosis, indicating that these two infection models provide different insights into the roles of Yops. To determine how wild-type Y. pseudotuberculosis hinders yop mutant survival, yop mutant colonization and host responses were investigated in several different infection models that isolated specific features of wild-type Y. pseudotuberculosis infection. Infection with wild-type Y. pseudotuberculosis caused significantly more inflammation than yop mutants. Results from coinfections of gamma interferon (IFN-gamma)-/- mice revealed that IFN-gamma-regulated defenses target these mutants, suggesting that YopE and YopH protect Y. pseudotuberculosis from these defenses in BALB/c mice. We developed an oral-intraperitoneal infection model to evaluate the effects of spleen and liver colonization by Y. pseudotuberculosis on yop mutants in the intestines. Spleen and liver infection increased inflammation and decreased yop mutant survival in the intestines, indicating that infection of these organs has consequences in intestinal tissues. Finally, competition infections with Y. pseudotuberculosis mutants with various abilities to induce inflammation demonstrated that survival of the yopE, but not the yopH, mutant was consistently decreased in inflamed tissues. In summary, infection with Y. pseudotuberculosis in intestinal and systemic sites induces intestinal inflammation, which decreases yop mutant survival. Thus, competition studies with wild-type yersiniae reveal critical roles of Yops in combating host responses to a normal virulent infection.

Administration, Oral↗

PubMed2006

Yersinia has a tropism for B and T cell zones of lymph nodes that is independent of the type III secretion system.

Pathogenic Yersinia have a pronounced tropism for lymphatic tissues and harbor a virulence plasmid that encodes a type III secretion system, pTTSS, that transports Yops into host cells. Yops are critical virulence factors that prevent phagocytosis by macrophages and neutrophils and Yersinia mutants lacking one or more Yops are defective for survival in lymphatic tissues, liver, and gastrointestinal tract. However, here we demonstrate that Y. pseudotuberculosis (Yptb) mutants lacking the pTTSS survived as well as or better than wild-type (WT) Yptb in the mesenteric lymph nodes (MLN). Infection with pTTSS mutants caused lymphadenitis with little necrosis, whereas infection with WT Yptb provoked lymphadenitis with multiple necrotic suppurative foci. Gentamicin protection assays and microscopic examination of the MLN revealed that pTTSS mutants resided extracellularly adjacent to B and T lymphocytes in the cortex and paracortex. WT Yptb was found extracellularly adjacent to neutrophils and macrophages in necrotic areas and adjacent to B and T lymphocytes in less-inflamed areas. To determine whether lymphocytes protected pTTSS mutants from phagocytic cells, Rag1(-/-) mice were infected with pTTSS mutants or WT Yptb. pTTSS mutants but not WT, were impaired for survival in MLN of Rag1(-/-) mice, suggesting that lymphocyte-rich regions constitute a protective niche for pTTSS mutants. Finally, we show that invasin and the chromosomally encoded TTSS were not required for Yptb survival in MLN. In summary, chromosomally encoded factors are sufficient for Yptb replication in the cortex and paracortex of MLN; the pTTSS enables Yersinia to survive within phagocyte-rich areas of lymph nodes, and spread to other tissues.

Adhesins, Bacterial↗

PubMed2004

Evolutionary genetics: CCR5 mutation and plague protection.

A recent and prevalent mutation in the chemokine receptor CCR5 in humans of northern European ancestry has been proposed to provide protection against bubonic plague. Here we infect both normal and CCR5-deficient mice with the bacterium Yersinia pestis, the cause of the plague epidemics that wiped out one-third of Europeans in the Middle Ages, and find no difference in either bacterial growth or survival time between the two groups. Unless the pathogenesis of Yersinia infection differs markedly between mice and humans, our results indicate that CCR5 deficiency in people is unlikely to protect against plague.

Animals↗

PubMed2003

Requirement of the Yersinia pseudotuberculosis effectors YopH and YopE in colonization and persistence in intestinal and lymph tissues.

The gram-negative enteric pathogen Yersinia pseudotuberculosis employs a type III secretion system and effector Yop proteins that are required for virulence. Mutations in the type III secretion-translocation apparatus have been shown to cause defects in colonization of the murine cecum, suggesting roles for one or more effector Yops in the intestinal tract. To investigate this possibility, isogenic yop mutant strains were tested for their ability to colonize and persist in intestinal and associated lymph tissues of the mouse following orogastric inoculation. In single-strain infections, a yopHEMOJ mutant strain was unable to colonize, replicate, or persist in intestinal and lymph tissues. A yopH mutant strain specifically fails to colonize the mesenteric lymph nodes, but yopE and yopO mutant strains showed only minor defects in persistence in intestinal and lymph tissues. While no single Yop was found to be essential for colonization or persistence in intestinal tissues in single-strain infections, the absence of both YopH and YopE together almost eliminated colonization of all tissues, indicating either that these two Yops have some redundant functions or that Y. pseudotuberculosis employs multiple strategies for colonization. In competition infections with wild-type Y. pseudotuberculosis, the presence of wild-type bacteria severely hindered the ability of the yopH, yopE, and yopO mutants to persist in many tissues, suggesting that the wild-type bacteria either fills colonization niches or elicits host responses that the yop mutants are unable to withstand.

Animals↗

PubMed2002

Use of signature-tagged mutagenesis in pathogenesis studies.

From 2000-2001, over 10 studies have been completed, using signature-tagged mutagenesis, on a variety of bacterial pathogens. Investigators are examining the attenuated strains in additional in vivo, cell culture or in vitro assays to further characterize and catalog the mutants. Notable advances included screening of the attenuated mutants en masse in other assays, verification that mutant strains are defective for growth in specific host tissues, and identification of potential live-attenuated vaccine candidates. In addition, researchers are using signature-tagged strains to characterize the infection process, which is providing snapshots of bottlenecks and quantitative measurements of bacterial spread from tissue to tissue. This review focuses on themes emerging from signature-tagged mutagenesis studies completed on bacterial pathogens since mid-2000.

Animals↗