Listeria monocytogenes wall teichoic acid decoration in virulence and cell-to-cell spread.

Wall teichoic acid (WTA) comprises a class of glycopolymers covalently attached to the peptidoglycan of gram positive bacteria. In Listeria monocytogenes, mutations that prevent addition of certain WTA decorating sugars are attenuating. However, the steps required for decoration and the pathogenic process interrupted are not well described. We systematically examined the requirement for WTA galactosylation in a mouse oral-virulent strain by first creating mutations in four genes whose products conferred resistance to a WTA-binding bacteriophage. WTA biochemical and structural studies indicated that galactosylated WTA was directly required for bacteriophage adsorption and that mutant WTA lacked appreciable galactose in all except one mutant - which retained a level ca. 7% of the parent. All mutants were profoundly attenuated in orally infected mice and were impaired in cell-to-cell spread in vitro. Confocal microscopy of cytosolic mutants revealed that all expressed ActA on their cell surface and formed actin tails with a frequency similar to the parent. However, the mutant tails were significantly shorter - suggesting a defect in actin based motility. Roles for the gene products in WTA galactosylation are proposed. Identification and interruption of WTA decoration pathways may provide a general strategy to discover non-antibiotic therapeutics for gram positive infections. © 2016 John Wiley & Sons Ltd.

Extrauterine listeriosis in the gravid mouse influences embryonic growth and development.

Gravid mice and other rodents inoculated with Listeria monocytogenes typically fail to clear an intrauterine infection and either succumb or expel their intrauterine contents. We took advantage of this property to investigate the effects of an extrauterine infection on parameters of pregnancy success. Pregnant mice were selected for our study if they showed no clinical signs of listeriosis following oral inoculation at 7.5 gestational days (gd), and had no detectable intrauterine colony forming units (cfu) at near term (18.5 gd). The range of oral doses employed was 106-108 cfu per mouse for two listerial serotype strains (4nonb and 1/2a). At all doses, inoculation resulted in a decrease in average near-term (18.5 gd) fetal weight per litter compared to sham inoculated controls. Additionally, embryonic death (indicated by intrauterine resorptions) was exhibited by some inoculated mice but was absent in all sham inoculated animals. In parallel experiments designed to detect possible loss of placental function, gravid uteruses were examined histopathologically and microbiologically 96 h after oral inoculation. Placental lesions were associated with high (> 106), but not low (< 10²) or absent intrauterine cfu. In vitro, mouse embryonic trophoblasts were indistinguishable from mouse enterocytes in terms of their sensitivity to listerial exposure. A model consistent with our observations is one in which products (host or bacterial) generated during an acute infection enter embryos transplacentally and influences embryonic survival and slows normal growth in utero.

In vitro properties of a Listeria monocytogenes bacteriophage-resistant mutant predict its efficacy as a live oral vaccine strain.

A Listeria monocytogenes glcV mutation precludes the binding of certain listerial phages and produces a profound attenuation characterized by the absence of detectable mutants in the livers and spleens of orally inoculated mice. In vitro, we found that the mutant formed plaques on mouse enterocyte monolayers as efficiently as the parent but the plaques formed were smaller. Intracellular growth rate determinations and examination of infected enterocytes by light and fluorescence microscopy established that the mutant was impaired not in intracellular growth rate but in cell-to-cell spreading. Because this property is shared by other immunogenic mutants (e.g., actA mutants), our glcV mutant was tested for vaccine efficacy. Oral immunization with the mutant and subsequent oral challenge (22 days postvaccination) with the parent revealed a ca. 10,000-fold increase in protection afforded by the mutant compared to sham-vaccinated controls. The glcV mutant did not stimulate innate immunity under the dose and route employed for vaccination, and an infectivity index time course experiment revealed pronounced mutant persistence in Peyer's patches. The immunogenicity of the glcV mutant compared to an isogenic actA mutant reference strain was next tested in an experiment with a challenge given 52 days postvaccination. Both mutant strains showed scant vital organ infectivity and high levels of protection similar to those seen using the glcV mutant in the 22-day postvaccination challenge. Our results indicate that oral administration of a profoundly attenuated listerial mutant can safely elicit solid protective immunity.

Faculty perceptions of appropriate faculty behaviors in social interactions with student pharmacists.

OBJECTIVE: To determine faculty and administrator perceptions about appropriate behavior in social interactions between pharmacy students and faculty members. METHODS: Four private and 2 public colleges and schools of pharmacy conducted focus groups of faculty members and interviews with administrators. Three scenarios describing social interactions between faculty members and students were used. For each scenario, participants reported whether the faculty member's behavior was appropriate and provided reasons for their opinions. RESULTS: Forty-four percent of those surveyed or interviewed considered interactions between faculty members and pharmacy students at a bar to be a boundary violation. Administrators were more likely than faculty members to consider discussing other faculty members with a student to be a boundary violation (82% vs. 46%, respectively, P <0.009). A majority (87%) of faculty members and administrators considered "friending" students on Facebook a boundary violation. CONCLUSIONS: There was no clear consensus about whether socializing with students at a bar was a boundary violation. In general, study participants agreed that faculty members should not initiate friendships with current students on social networks but that taking a student employee to lunch was acceptable.

Factors associated with the acquisition and severity of gestational listeriosis.

Gravid mammals are more prone to listeriosis than their nongravid counterparts. However, many features of the disease in gravid animals are not well defined. We determined, in mice, that increased susceptibility to lethal infection following oral inoculation begins surprisingly early in pregnancy and extends through embryonic development. Pregnancy did not demonstrably increase the spread of listeriae from the intestine to the liver and spleen in the initial 96 h period post inoculation. Consequently, it appeared that gravid animals were competent to contain an enteric infection, but in those instances where escape did occur, a lethal outcome was more likely. Interestingly, colonic colonization level and prevalence, measured 96 h post inoculation, was significantly higher in gravid individuals. In terms of human risk factors for listeriosis, our results suggest that the window of listeriosis susceptibility afforded by pregnancy may be open longer than previously appreciated. Our results also suggest that while gravid animals are competent to contain an enteric infection, enteric carriage rate may be more of a factor in defining disease incidence than previously considered.

A Listeria monocytogenes mutant defective in bacteriophage attachment is attenuated in orally inoculated mice and impaired in enterocyte intracellular growth.

A Listeria monocytogenes bacteriophage was used to identify a phage-resistant Tn917 insertion mutant of the mouse-virulent listerial strain F6214-1. The mutant was attenuated when it was inoculated orally into female A/J mice and failed to replicate efficiently in cultured mouse enterocytes. Phage binding studies indicated that the mutant had a cell surface alteration that precluded phage attachment. All phenotypes associated with the mutation could be complemented in trans by a single open reading frame (ORF) that corresponded to the ORF interrupted by the Tn917 insertion. The complementation effected was, in all cases, at a level indistinguishable from that of the parent. The Tn917 insertion interrupted a gene that is predicted to encode a group 2 glycosyl transferase (provisionally designated glcV). A similar glcV gene is present in Listeria welshimeri and Listeria innocua and in some serotypes of L. monocytogenes. We speculate that the loss of the glcV product results in a defective phage receptor and that this alteration coincidentally influences a feature of the normal host-pathogen interaction required for virulence. Interestingly, the glcV lesion, while preventing phage attachment, did not alter the mutant's ability to bind to cultured mouse enterocyte monolayers. Rather, the mutation appeared to alter a subsequent step in intracellular replication measured by a reduction in plaque-forming efficiency and plaque size. In vivo, the mutant was undetectable in the liver and spleen 48 h after oral inoculation. The mutation is significant in part because it is one of the few that produce attenuation when the mutant is delivered orally.

Host and bacterial factors in listeriosis pathogenesis.

Members of the Genus Listeria are ubiquitous environmental saprophytic microorganisms. If ingested they can cause a severe disseminated disease (listeriosis) that has a high mortality rate, the highest of any food-borne pathogen, even with antibiotic therapy. Central to the high mortality rate is the hallmark characteristic of the microorganism to grow intracellularly. The presence of listeriae in food processing plants has resulted in many outbreaks of human disease and large scale recalls of processed foods. Despite the ubiquity of the microorganism, the actual disease rate (those animals showing disease signs over those exposed) is quite low and disease is almost always associated with an underlying predisposition (pregnancy being the most common in otherwise normal individuals). There are many features of the pathogenesis of listeriosis that have remained mysterious despite the extensive use of the microorganism in the study of cell-mediated immunity and intracellular growth. Informational advances such as the sequence of the mouse and listerial genomes, and technical advances such as the discovery of listeria-susceptible mouse strains, may renew interest in the study of the natural pathogenesis of the disease. This may be further facilitated by studies that employ the natural inoculation route and mimic common predisposing conditions witnessed in victims of natural outbreaks.

Influence of pregnancy on the pathogenesis of listeriosis in mice inoculated intragastrically.

Pregnancy increases the risk of listeriosis, a systemic disease caused by Listeria monocytogenes. However, there is incomplete agreement on the reasons for this increased risk. We examined two features of listeriosis in gravid and nongravid female mice following intragastric (gavage) inoculation, namely, (i) disease severity (measured by lethality) and (ii) listerial infectivity (measured by liver and spleen colonization levels up to 120 h postinoculation). Two listerial strains of differing serotype (1/2a and 4nonb) were initially employed. Neither strain produced a lethal infection in nonpregnant female mice (dose range, 10(6) to 10(9) CFU/mouse), and only the 4nonb strain produced lethalities in pregnant mice (dose range, 10(6) to 10(8) CFU/mouse). The 4nonb strain also produced a higher level of liver and spleen colonization than the 1/2a strain following gavage administration. (The two strains showed similar levels of colonization if parenterally administered.) Both strains were equally capable of binding to and forming plaques upon cultured mouse enterocytes. The ability of the 4nonb strain to produce a lethal infection in pregnant animals did not correlate with an increased incidence or level of liver and spleen colonization over that in nonpregnant females. However, the lethality rate did correlate well with the rate at which embryos and their surrounding decidual covering became infected, suggesting that intrauterine infection could be responsible for the increased disease severity in the gravid females.

Influence of extracellular bactericidal agents on bacteria within macrophages.

We employed gentamicin-sensitive and -resistant derivatives of Escherichia coli in a macrophage phagocytosis assay that compared lambda bacteriophage and gentamicin as extracellular bactericidal agents. Colony counts and direct microscopic examination of phagocytized E. coli supported the conclusion that gentamicin entered macrophages, even at low concentrations, and contributed to their bactericidal activity. Also, two E. coli strains differing in the ability to express the adhesin of type 1 pili (FimH) were distinguishably different in intracellular survival when lambda was used as the extracellular killing agent but were indistinguishable when gentamicin was employed.

Antigenic variation of gonococcal pilin expression in vivo: analysis of the strain FA1090 pilin repertoire and identification of the pilS gene copies recombining with pilE during experimental human infection.

Antigenic variation of gonococcal pilin involves a family of variable genes that undergo homologous recombination, resulting in transfer of variant sequences from the pilS silent gene copies into the complete pilE expression locus. Little is known about the specific recombination events that are involved in assembling new variant pilin genes in vivo. One approach to understanding pilin variation in vivo is to carry out experimental human infections with a gonococcal strain having a fully characterized repertoire of pilin genes, so that the specific recombination events occurring in vivo can be determined. To this end, the authors cloned, sequenced and mapped the pilin genes of strain FA1090 of Neisseria gonorrhoeae. This strain contains one pilE locus and 19 silent gene copies that are arranged in five pilS loci; the pilE locus and four of the pilS loci are clustered in a 35 kb region of the chromosome. The general features of the pilin loci in FA1090 are similar to those in strain MS11, in which the mechanism of pilin variation has been extensively studied. However, none of the silent copy sequences are identical in the two strains, which emphasizes the extreme variability in this gene family among gonococci. Three male volunteers were inoculated with the same variant of strain FA1090 and developed urethritis within 2--4 d. The pilE gene sequences from a total of 23 colonies cultured from the subjects were analysed, determining which pilS silent copy donated each portion of the expressed pilE genes. There were 12 different pilin variants, one of which was the original inoculum variant, among the in vivo-expressed pilE gene sequences. The pilE of the inoculum variant was derived entirely from a single silent copy (pilS6c1). However, the pilE genes in the majority of the colonies cultured from the infected subjects were chimeras of sequence derived from two or three silent copies. Recombination to generate new pilE sequences involved exchange of single variable minicassettes, multiple minicassettes, entire silent gene copies, or (rarely) recombination within a minicassette.