John R. Horton

Characterization of Escherichia coli Type 1 Pilus Mutants with Altered Binding Specificities

PCR mutagenesis and a unique enrichment scheme were used to obtain two mutants, each with a single lesion in fimH, the chromosomal gene that encodes the adhesin protein (FimH) of Escherichia coli type 1 pili. These mutants were noteworthy in part because both were altered in the normal range of cell types bound by FimH. One mutation altered an amino acid at a site previously shown to be involved in temperature-dependent binding, and the other altered an amino acid lining the predicted FimH binding pocket.

Host and Bacterial Factors Involved in the Innate Ability of Mouse Macrophages To Eliminate Internalized Unopsonized Escherichia coli

ABSTRACT In an effort to better understand genetic and cellular factors that influence innate immunity, we examined host and bacterial factors involved in the nonopsonic phagocytosis and killing ofEscherichia coli K-12 by mouse macrophages. Unelicited (resident) peritoneal macrophages from five different mouse strains, BALB/c, C57BL/6, CD-1, C3H/HeJ, and C3H/HeN, were employed. Additional macrophage populations were obtained from CD-1 mice (bone marrow-derived macrophages). Also, for BALB/c and C57BL/6 mice, peritoneal macrophages elicited with either thioglycolate or proteose peptone, bone marrow-derived macrophages, and macrophage-like cell lines derived from the two strains were employed. Two E. coli K-12 strains that differed specifically in their abilities to produce type 1 pili containing the adhesive protein FimH were examined. The parameters used to assess macrophage bacteriocidal activity were (i) the killing of internalized (gentamicin-protected)E. coli during the approximately 4-h assay and (ii) the initial rate at which internalized E. coli were eliminated. Data on these parameters allowed the following conclusions: (i) unelicited or proteose peptone-elicited peritoneal macrophages were significantly better at eliminating internalized bacteria than thioglycolate-elicited peritoneal macrophages, bone marrow-derived macrophages, or macrophage cell lines; (ii) the host genetic background had no significant effect upon the ability of unelicited peritoneal macrophages to kill E. coli (even though the mouse strains differ widely in their in vivo susceptibilities to bacterial infection); and (iii) the FimH phenotype had no significant effect uponE. coli survival once the bacterium was inside a macrophage. Additionally, there was no correlation between the bacteriocidal effectiveness of a macrophage population and the number of bacteria bound per macrophage. However, macrophage populations that were the least bacteriocidal tended to bind higher ratios of FimH+ to FimH−E. coli. The effect of gamma interferon, fetal calf serum, and the recombination proficiency of E. coli were examined as factors predicted to influence intracellular bacterial killing. These had no effect upon the rate of E. coli elimination by unelicited peritoneal macrophages.

Influence of extracellular bactericidal agents on bacteria within macrophages.

ABSTRACT We employed gentamicin-sensitive and -resistant derivatives of Escherichia coli in a macrophage phagocytosis assay that compared λ 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 λ was used as the extracellular killing agent but were indistinguishable when gentamicin was employed.

Influence of Pregnancy on the Pathogenesis of Listeriosis in Mice Inoculated Intragastrically

ABSTRACT 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, 106 to 109 CFU/mouse), and only the 4nonb strain produced lethalities in pregnant mice (dose range, 106 to 108 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.

Formation of antibiotic, biodegradable/bioabsorbable polymers by processing with neomycin sulfate and its inclusion compound with β‐cyclodextrin

Samples of pure neomycin sulfate and its inclusion compound (IC) with β-cyclodextrin were implanted into films of poly(L-lactic acid) (PLLA) and poly(e-caprolactone) (PCL). Both polymers have been widely used commercially to make sutures. The antibacterial activity of these films against Escherichia coli was tested. Films made by either solution casting or melt pressing were divided into the following three groups: (1) plain polymer films, (2) those embedded with pure neomycin sulfate, and (3) those embedded with neomycin sulfate-β-cyclodextrin IC. Filter paper treated with 1.5 μL of 10 mg/μL Kanamycin and neomycin were used as controls and resulted in 11- and 8-mm zones of inhibition/or antibacterial activity, respectively. Small discs (ca. 2% of total area) cut from solution-cast films of PLLA and PCL containing 50 wt % neomycin sulfate IC had 17- and 16-mm zones of inhibition, and PLLA and PCL containing 50 wt % pure neomycin sulfate deterred bacterial growth, resulting in 19-mm zones of inhibition. Melt-pressed films containing 10 wt % pure neomycin sulfate or its IC, showed 17- and 11-mm zones of inhibition for PLLA films, respectively, while PCL films showed 13- and 9-mm zones of inhibition, respectively. For melt-pressed films that contain 0.01 wt % pure neomycin sulfate or its IC, PLLA films showed 11- and 9.5-mm zones of inhibition, respectively, while PCL films showed 11- and 10-mm zones of inhibition, respectively. Since an antibiotic, bioabsorbable suture does not require surgical removal, implanting an inclusion compound in the suture might allow the slow release of antibiotic, thereby guarding against postsurgical infection and also protecting the antibiotic from degradation during the melt-spinning process used to make the suture.

A Listeria monocytogenes Mutant Defective in Bacteriophage Attachment Is Attenuated in Orally Inoculated Mice and Impaired in Enterocyte Intracellular Growth

ABSTRACT 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 mutants 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.

Identification and Characterization of Two Bordetella avium Gene Products Required for Hemagglutination

ABSTRACT Bordetella avium causes bordetellosis in birds, a disease similar to whooping cough caused by Bordetella pertussis in children. B. avium agglutinates guinea pig erythrocytes via an unknown mechanism. Loss of hemagglutination ability results in attenuation. We report the use of transposon mutagenesis to identify two genes required for hemagglutination. The genes (hagA and hagB) were adjacent and divergently oriented and had no orthologs in the genomes of other Bordetella species. Construction of in-frame, unmarked mutations in each gene allowed examination of the role of each in conferring erythrocyte agglutination, explanted tracheal cell adherence, and turkey poult tracheal colonization. In all of the in vitro and in vivo assays, the requirement for the trans-acting products of hagA and hagB (HagA and HagB) was readily shown. Western blotting, using antibodies to purified HagA and HagB, revealed proteins of the predicted sizes of HagA and HagB in an outer membrane-enriched fraction. Antiserum to HagB, but not HagA, blocked B. avium erythrocyte agglutination and explanted turkey tracheal ring binding. Bioinformatic analysis indicated the similarity of HagA and HagB to several two-component secretory apparatuses in which one product facilitates the exposition of the other. HagB has the potential to serve as a useful immunogen to protect turkeys against colonization and subsequent disease.

Genetic Characterization of Escherichia coli Type 1 Pilus Adhesin Mutants and Identification of a Novel Binding Phenotype

Five Escherichia coli type 1 pilus mutants that had point mutations in fimH, the gene encoding the type 1 pilus adhesin FimH, were characterized. FimH is a minor component of type 1 pili that is required for the pili to bind and agglutinate guinea pig erythrocytes in a mannose-inhibitable manner. Point mutations were located by DNA sequencing and deletion mapping. All mutations mapped within the signal sequence or in the first 28% of the predicted mature protein. All mutations were missense mutations except for one, a frameshift lesion that was predicted to cause the loss of approximately 60% of the mature FimH protein. Bacterial agglutination tests with polyclonal antiserum raised to a LacZ-FimH fusion protein failed to confirm that parental amounts of FimH cross-reacting material were expressed in four of the five mutants. The remaining mutant, a temperature-sensitive (ts) fimH mutant that agglutinated guinea pig erythrocytes after growth at 31 degrees C but not at 42 degrees C, reacted with antiserum at both temperatures in a manner similar to the parent. Consequently, this mutant was chosen for further study. Temperature shift experiments revealed that new FimH biosynthesis was required for the phenotypic change. Guinea pig erythrocyte and mouse macrophage binding experiments using the ts mutant grown at the restrictive and permissive temperatures revealed that whereas erythrocyte binding was reduced to a level comparable to that of a fimH insertion mutant at the restrictive temperature, mouse peritoneal macrophages were bound with parental efficiency at both the permissive and restrictive temperatures. Also, macrophage binding by the ts mutant was insensitive to mannose inhibition after growth at 42 degrees C but sensitive after growth at 31 degrees C. The ts mutant thus binds macrophages with one receptor specificity at 31 degrees C and another at 42 degrees C.

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.