bluuebunny/crossref_metadata_2025 huggingface dataset:
Deletion of
porA
by Recombination between Clusters of Repetitive Extragenic Palindromic Sequences in
Neisseria meningitidis
Authors: van der Ende A. , Hopman C. T. P. , Dankert J.
Published: December 2019

PorA is an important component in a vaccine against infection with
Neisseria meningitidis
. However,
porA
-negative meningococci were isolated from patients, thereby potentially limiting the role of PorA-mediated immunity. To analyze the mechanism by which the
porA
deletion occurred, the regions upstream and downstream of
porA
from three meningococcal strains (H44/76, H355, and 860183) were sequenced. The
porA
upstream region in strain 860183 contains a cluster of 22 repetitive palindromic RS3 core sequences (ATTCCC-N
8
-GGGAAT) and 10 RS3 core sequences (ATTCCC) in direct orientation. The cluster is flanked by neisserial repeats, so-called Correia elements, and can be subdivided into three repeats of 518 bp followed by a truncated repeat. The
porA
upstream region of the other two strains showed deletions, probably caused by a recombination between RS3 core sequences. The
porA
downstream region of H44/76 and H355 contains the IS
1106
element followed by a cluster of 10 palindromic RS3 core sequences, 4 RS3 core sequences, and 1 other RS3 core sequence (GGGAAT) and is followed by a Correia element. This cluster can be subdivided into four direct repeats of 370 bp. Strain 860183 had two such repeats instead of four. Sequence analysis of the
porA
-negative variants indicated that the deletion of
porA
occurred via a recombination between two copies of the 116-bp region, containing two palindromic RS3 core sequences and a single RS3 core sequence. This region is homologous in the upstream and downstream clusters.
Lipid Extract ofMycoplasma penetransProteinase K-Digested Lipid-Associated Membrane Proteins Rapidly Activates NF-κB and Activator Protein 1
Authors: Feng Shaw-Huey , Lo Shyh-Ching
Published: December 2019

Lipid-associated membrane proteins (LAMPs) ofMycoplasma penetransrapidly induced macrophages to produce proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α). Our analysis showed that the macrophage-stimulating activity of TNF-α production was mainly attributable to a lipid extractable component(s) in the LAMP preparation. Since induction of gene expression is normally preceded by activation of transcriptional factors that bind to their specific recognition elements located in the upstream promoter region, we examined the activity of transcriptional factors, namely, NF-κB and activator protein 1 (AP-1), in thioglycolate exudate peritoneal (TEP) macrophages treated withM. penetranslipid extract of proteinase K (PK)-digested LAMPs. Initially, in the nuclei of unstimulated TEP cells, there was only a low basal level of active AP-1, and the active form of NF-κB could not be detected.M. penetranslipid extract of PK-digested LAMPs activated both NF-κB and AP-1 in TEP macrophages within 15 min. The markedly increased activities of both factors gradually declined and dissipated after 2 h. Parallel to the rapid increase of NF-κB and AP-1, the TNF-α transcript also increased significantly 15 min after the stimulation. The high-level expression of TNF-α persisted over 2 h. Dexamethasone blocked the activation of both NF-κB and AP-1 and suppressed the production of TNF-α in TEP macrophages stimulated byM. penetranslipid extract of PK-digested LAMPs. Our study demonstrates that theM. penetranslipid extract of PK-digested LAMP is a potent activator for NF-κB and AP-1 in murine TEP macrophages. Our results also suggest that high-level expression of TNF-α in cells induced byM. penetranslipid extract of PK-digested LAMPs is associated with rapid activation of transcriptional factors NF-κB and AP-1.
Prior Genital Tract Infection with a Murine or Human Biovar ofChlamydia trachomatisProtects Mice against Heterotypic Challenge Infection
Authors: Ramsey Kyle H. , Cotter Todd W. , Salyer Rena D. , Miranpuri Gurwattan S. , Yanez Michael A. , Poulsen Christoffer E. , DeWolfe Jennifer L. , Byrne Gerald I.
Published: December 2019

We sought to assess the degree of cross-protective immunity in a mouse model of chlamydial genital tract infection. Following resolution of genital infection with the mouse pneumonitis (MoPn) biovar ofChlamydia trachomatis, mice were challenged intravaginally with either MoPn or human serovar E or L2. The majority of animals previously infected with MoPn were solidly immune to challenge with either of the two human biovars. Surprisingly, approximately 50% of animals became reinfected when homologously challenged with MoPn, although the secondary infection yielded significantly lower numbers of the organism isolated over a shorter duration than in the primary infection. Primary infection with serovar E also protected against challenge with MoPn or serovar L2, although the degree of immune protection was lower than that resulting from primary infection with MoPn. Blast transformation and assessment of delayed-type hypersensitivity indicated that mice previously infected with either human or murine biovars produced broadly cross-reactive T cells that recognized epitopes of either murine or human biovars ofC. trachomatis. Immunoblotting demonstrated that primary MoPn infection produced immunoglobulin G (IgG) antibody to antigens of MoPn as well as at least three distinct antigenic components of human serovar E, one of which was identical in molecular weight to the major outer membrane protein (MOMP). Primary infection with serovar E produced IgG antibody reactive against serovar E but not MoPn MOMP and against at least one ca. 60-kDa protein of both chlamydial strains. Our results indicate that primary genital infection of mice with murineC. trachomatisinduces immunity against challenge with either of two human biovars.
Porcine Epithelial β-Defensin 1 Is Expressed in the Dorsal Tongue at Antimicrobial Concentrations
Authors: Shi Jishu , Zhang Guolong , Wu Hua , Ross Christopher , Blecha Frank , Ganz Tomas
Published: December 2019

Epithelial cells and phagocytes contain antimicrobial polypeptides that participate in innate host defense. A recently cloned porcine β-defensin, PBD-1, was detected by Northern organ blots exclusively in the tongue epithelium. We generated recombinant PBD-1 peptide by using a baculovirus-insect cell expression system and obtained two forms (PBD-1
42
and PBD-1
38
), which differed by N-terminal truncation. Only PBD-1
42
was found in scrapings of the surface of the dorsal tongue or the buccal mucosa. Immunohistochemical staining with antibody to PBD-1
42
revealed that PBD-1 was highly concentrated in an ∼0.1-mm-thick layer in the cornified tips of the filiform (but not fungiform) papillae of the dorsal tongue and in the superficial squamous cell layers of the buccal mucosa. By scraping, extraction, and semiquantitative Western blotting, the concentration of PBD-1 in the dorsal tongue surface and the buccal mucosa was estimated at 20 to 100 μg/ml. PBD-1 had antibacterial activity against
Escherichia coli
,
Salmonella typhimurium
,
Listeria monocytogenes
, and
Candida albicans
in 10 mM sodium phosphate buffer (pH 7.4). Added NaCl progressively inhibited the activity of PBD-1 against
E. coli
and
C. albicans
. In 10 mM sodium phosphate with 125 mM NaCl, the combinations of sublethal concentrations of PBD-1 and the porcine neutrophil peptide PG-3, PR-39, or PR-26 showed synergistic activity against
E. coli
or the multidrug-resistant
S. typhimurium
DT104. At its physiologic concentration, PBD-1 has antimicrobial effects under both low- and high-salt conditions encountered in the oral cavity and may contribute to the antimicrobial barrier properties of the dorsal tongue and oral epithelium.
FbpC Is Not Essential for Iron Acquisition in
Neisseria gonorrhoeae
Authors: Sebastian Shite , Genco Caroline Attardo
Published: December 2019

The
fbpABC
locus of
Neisseria gonorrhoeae
has been proposed to encode a periplasmic protein-dependent iron transport system. Although the function of the gonococcal FbpA protein has been well characterized and its role as a periplasmic binding protein is well defined, little is known about the function of the FbpB and FbpC proteins. To define the function of the gonococcal FbpC protein, an
N. gonorrhoeae
F62
fbpC
mutant was constructed by insertional inactivation with the kanamycin gene. The
N. gonorrhoeae
F62
fbpC
mutant was observed to grow with heme, transferrin, or ferric nitrate as the sole exogenous iron source, indicating that the gonococcal FbpC protein is not absolutely required for growth with these iron sources. In previous studies we were unable to detect
fbpB
- or
fbpC
-specific transcripts by Northern analysis. Reverse transcription-PCR analysis with RNA obtained from
N. gonorrhoeae
F62 grown under iron-replete and -depleted conditions detected
fbpA
and
fbpAB
transcripts but failed to detect
fbpC
or
fbpBC
transcripts. These results indicate that FbpC does not play a pivotal role in iron transport in
N. gonorrhoeae
and suggest that additional ABC transport systems are functional in the gonococcus for the acquisition of iron.
Early Gene Expression of NK Cell-Activating Chemokines in Mice Resistant toLeishmania major
Authors: Vester Bettina , Müller Kerstin , Solbach Werner , Laskay Tamás
Published: December 2019

Susceptibility of mice toLeishmania majoris associated with an insufficient NK cell-mediated innate immune response. We analyzed the expression of NK cell-activating chemokines in vivo during the first days of infection in resistant and susceptible mice. The mRNA expression of gamma interferon-inducible protein 10 (IP-10), monocyte chemoattractant protein 1 (MCP-1), and lymphotactin was upregulated 1 day after infection in the draining lymph nodes of resistant C57BL/6 mice but not in those of susceptible BALB/c mice. In vivo local treatment of BALB/c mice with recombinant IP-10 shortly after infection resulted in an enhanced NK cell activity in the draining lymph node. The data suggest that although the recruitment of NK cells is normal in susceptible mice, the lack of NK cell-activating chemokines is a factor resulting in a suboptimal NK cell-mediated defense.
Immunity in Experimental Murine Filariasis: Roles of T and B Cells Revisited
Authors: Babu Subash , Shultz Leonard D. , Klei Thomas R. , Rajan T. V.
Published: December 2019

We have reevaluated the contributions of T and B cells inBrugia malayiinfection by utilizing knockout mice on a uniform background (C57BL/6J). We find that B-cell-deficient mice are more permissive to infection than T-cell-deficient mice.
Effects of
Mycoplasma fermentans
incognitus on Differentiation of THP-1 Cells
Authors: Reyes Leticia , Davidson Maureen K. , Thomas Linda C. , Davis Jerry K.
Published: December 2019

Mycoplasma fermentans
incognitus has been isolated from human tissue in patients both with and without AIDS who died of systemic infection.
M. fermentans
incognitus and other strains of
M. fermentans
have been associated with rheumatoid arthritis. While cell extracts of
M. fermentans
incognitus can induce changes in murine and human cells of the monocytic lineage, little is known about interactions of viable organisms with such cells. Because of the central role of macrophages in chronic inflammation, we examined the effects of
M. fermentans
incognitus on surface markers and functions of THP-1 cells, a well-characterized human monocytic cell line. This cell line has been used extensively in studies of macrophage differentiation, especially following exposure to phorbol esters. Changes in cell morphology, phagocytosis, rate of cell division, and selected surface markers were evaluated in cultures of THP-1 cells exposed to phorbol myristate acetate (PMA),
M. fermentans
incognitus, or both. As reported by other investigators, PMA induced THP-1 cells to differentiate into cells resembling tissue macrophages.
M. fermentans
incognitus only minimally affected changes induced by PMA, slightly increasing the percentage of cells positive for FCγRI and major histocompatibility complex (MHC) class II antigens.
M. fermentans
incognitus alone induced an incomplete arrest in the cell cycle at G
0
phase, increased phagocytic ability, and enhanced expression of FCγRI, CR3, CR4, and MHC class II antigens.
Survival ofMycobacterium aviumandMycobacterium tuberculosisin Acidified Vacuoles of Murine Macrophages
Authors: Gomes Maria Salomé , Paul Simon , Moreira Andre L. , Appelberg Rui , Rabinovitch Michel , Kaplan Gilla
Published: December 2019

Despite the antimicrobial mechanisms of vertebrate phagocytes, mycobacteria can survive within the phagosomes of these cells. These organisms use various strategies to evade destruction, including inhibition of acidification of the phagosome and inhibition of phagosome-lysosome fusion. In contrast to mycobacteria,Coxiella burnetii, the etiologic agent of Q fever, inhabits a spacious acidified intracellular vacuole which is prone to fusion with other vacuoles of the host cell, including phagosomes containing mycobacteria. TheCoxiella-infected cell thus provides a unique model for investigating the survival of mycobacteria in an acidified phagosome-like compartment. In the present study, murine bone marrow-derived macrophages were infected with eitherMycobacterium aviumorMycobacterium tuberculosisand then coinfected withC. burnetii. We observed that the majority of phagocytosed mycobacteria colocalized to theC. burnetii-containing vacuole, which maintained its acidic properties. In coinfected macrophages, the growth ofM. aviumwas not impaired following fusion with the acidified vacuole. In contrast, the growth rate ofM. tuberculosiswas reduced in acidified vacuoles. These results suggest that although both species of mycobacteria inhibit phagosome-lysosome fusion, they may be differentially susceptible to the toxic effects of the acidic environment in the mature phagolysosome.
Innate Antimicrobial Activity of Nasal Secretions
Authors: Cole Alexander M. , Dewan Puneet , Ganz Tomas
Published: December 2019

Minimally manipulated nasal secretions, an accessible form of airway surface fluid, were tested against indigenous and added bacteria by using CFU assays. Antimicrobial activity was found to vary between donors and with different target bacteria and was markedly diminished by dilution of the airway secretions. Donor-to-donor differences in electrophoresis patterns of nasal secretions in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) and acid urea-PAGE analyses were readily observed, suggesting that polymorphic genes encode the secreted proteins. Three donors (of twenty-four total), whose nasal fluid yielded similar protein band patterns and did not kill indigenous bacteria, were determined to be heavy nasal carriers of
Staphylococcus aureus
. Their fluid was deficient in microbicidal activity toward a colonizing strain of
S. aureus
but the defect was corrected in vitro by a 1:1 addition of nasal fluid from noncarriers. The microbicidal activity of normal fluid was inactivated by heating it for 10 min to 100°C and could not be restored solely by the addition of two major nasal antimicrobial proteins, lysozyme and lactoferrin. Several other known antimicrobial proteins and peptides, including statherin, secretory phospholipase A
2
, and defensins, were identified in nasal secretions and likely contribute to their total antimicrobial properties. Nasal fluid may serve as a useful model for the analysis of lower-airway secretions and their role in host defense against airway colonization and pulmonary infections.
Role of Lactosyl Glycan Sequences in Inhibiting Enteropathogenic
Escherichia coli
Attachment
Authors: Vanmaele Rosa P. , Heerze Louis D. , Armstrong Glen D.
Published: December 2019

Previously, we found that asialo-lactosamine sequences served as receptors for enteropathogenic
Escherichia coli
(EPEC) binding to Chinese hamster ovary (CHO) cells. In the present report, we have extended these earlier results by examining the ability of lactosamine- or fucosylated lactosamine-bovine serum albumin (BSA) glycoconjugates to inhibit EPEC, strain E2348/69, binding to HEp-2 cells. We found that, consistent with our previous findings with CHO cells,
N
-acetyllactosamine-BSA was the most effective inhibitor of EPEC localized adherence to HEp-2 cells, with Lewis X-BSA being the next best inhibitor. Further investigation revealed that coincubating EPEC E2348/69 with these BSA glycoconjugates alone caused a decrease in the expression of the bundle-forming pilus structural subunit (BfpA) and intimin by the bacteria. BfpA and intimin expression were reduced to the greatest extent by
N
-acetyllactosamine–BSA and Lewis X-BSA, respectively. These results suggest that the glycoconjugate inhibition of EPEC binding to HEp-2 cells might be achieved, wholly or in part, by an active mechanism that is distinct from simple competitive antagonism of receptor-adhesin interactions.
T-Cell Responses duringTrypanosoma bruceiInfections in Mice Deficient in Inducible Nitric Oxide Synthase
Authors: Millar Amanda E. , Sternberg Jerry , McSharry Charlie , Wei Xiao-Qing , Liew F. Y. , Turner C. Michael R.
Published: December 2019

We have investigated the possibility that nitric oxide (NO) synthesis may affect the course of a trypanosome infection via T-cell responses using mice deficient in inducible NO synthase (iNOS). Parasitemia levels increased at the same rate in both iNOS-deficient homozygous and control heterozygous mice, and peak parasitemia values were the same in both groups. However, the heterozygous mice maintained higher parasitemia levels after the peak of an infection than the homozygous mice due to a decrease in the rate of clearance of parasites. In iNOS-deficient mice there was an increase in the numbers of total CD4+cells and activated (interleukin-2 receptor-expressing) CD4+cells in infected mice compared with the numbers in uninfected mice. Spleen cells from infected iNOS-deficient mice displayed increased proliferative responses and gamma interferon secretion when stimulated in vitro than those of control mice. These data suggest that NO production depresses T-helper 1-like responses generated duringTrypanosoma bruceiinfections, thus promoting the survival of the parasite.
Pathogenesis of Gram-Positive Bacterial Endophthalmitis
Authors: Callegan Michelle C. , Booth Mary C. , Jett Bradley D. , Gilmore Michael S.
Published: December 2019

The severity of endophthalmitis has been associated generally with the virulence of the offending pathogen. However, precisely what constitutes the virulence in intraocular infections remains ill defined. We therefore sought to identify the basis for virulence for three common ocular pathogens (Bacillus cereus,Enterococcus faecalis, andStaphylococcus aureus) in terms of intraocular growth rates, bacterial localization patterns, and the contribution of cell walls and secreted products to the pathogenesis of endophthalmitis. Rabbit eyes were injected intravitreally with (i) viableB. cereus,E. faecalis, orS. aureus, (ii) metabolically inactiveB. cereus,E. faecalis, orS. aureus, (iii) sacculus preparations from each strain, or (iv) culture fluid containing products secreted by each strain. Eyes were assessed at various times following injection by slit lamp biomicroscopy, electroretinography (ERG), bacterial and inflammatory cell enumeration, and histology.B. cereusendophthalmitis followed a more rapid and virulent course thanE. faecalisorS. aureusendophthalmitis, eliminating retinal responsiveness, as measured by ERG, by 12 h. Analysis of bacterial localization revealed thatB. cereusuniquely migrated rapidly from posterior to anterior segment during infection. Although injection of neither metabolically inactive bacteria nor cell wall sacculi greatly affected ERG, significant intraocular inflammation was observed. Injection ofB. cereusorS. aureusculture fluids caused both significant reductions in retinal responsiveness and significant intraocular inflammation, paralleling that seen in natural infections. The results demonstrate that toxins, intraocular localization, and, to a lesser extent, the intraocular host response to cell walls all contribute to the pathogenesis ofB. cereus,S. aureus, andE. faecalisendophthalmitis in a pathogen-specific manner. The key pathophysiologic differences in these intraocular diseases highlight opportunities for optimizing conventional therapies and deriving new ones.
Deacylation of Purified Lipopolysaccharides by Cellular and Extracellular Components of a Sterile Rabbit Peritoneal Inflammatory Exudate
Authors: Weinrauch Yvette , Katz Seth S. , Munford Robert S. , Elsbach Peter , Weiss Jerrold
Published: December 2019

The extent to which the mammalian host is capable of enzymatic degradation and detoxification of bacterial lipopolysaccharides (LPS) is still unknown. Partial deacylation of LPS by the enzyme acyloxyacyl hydrolase (AOAH) provides such a mechanism, but its participation in the disposal of LPS under physiological conditions has not been established. In this study, deacylation of isolated radiolabeled LPS by both cellular and extracellular components of a sterile inflammatory peritoneal exudate elicited in rabbits was examined ex vivo. AOAH-like activity, tested under artificial conditions (pH 5.4, 0.1% Triton X-100), was evident in all components of the exudate (mononuclear cells [MNC] > polymorphonuclear leukocytes [PMN] > inflammatory [ascitic] fluid [AF]). Under more physiological conditions, in a defined medium containing purified LPS-binding protein, the LPS-deacylating activity of MNC greatly exceeded that of PMN. In AF, MNC (but not PMN) also produced rapid and extensive CD14-dependent LPS deacylation. Under these conditions, almost all MNC-associated LPS underwent deacylation within 1 h, a rate greatly exceeding that previously found in any cell type. The remaining extracellular LPS was more slowly subject to CD14-independent deacylation in AF. Quantitative analysis showed a comparable release of laurate and myristate but no release of 3-hydroxymyristate, consistent with an AOAH-like activity. These findings suggest a major role for CD14+MNC and a secondary role for AF in the deacylation of cell-free LPS at extravascular inflammatory sites.
Isolation of T-Cell Antigens by Using a Recombinant Protein Library and Its Application to the Identification of Novel Vaccine Candidates against Schistosomiasis
Authors: Eberl Matthias , Mountford Adrian P. , Jankovic Dragana , Beck Ewald
Published: December 2019

We present here a novel approach to identify T-cell antigens from any infectious agent by use of a library of purified recombinant proteins. Essential features of this strategy include (i) a highly efficient cDNA cloning system which negatively selects against nonrecombinant transformants by making use of the bacterialEcoK restriction system, (ii) affinity staining of cDNA clones expressing recombinant proteins, and (iii) a procedure of simultaneous purification of recombinant proteins from large numbers of isolated clones (representing the protein library) in a single step from pools consisting of up to 24 individual clones. The feasibility of the screening system was confirmed by constructing a protein library of the human parasiteSchistosoma mansoni. The recombinant antigens of this library were used to stimulate CD4+T cells derived from the axillary lymph nodes of mice vaccinated with irradiated cercariae. In initial screening experiments, we detected parasite-specific proliferation and gamma interferon (IFN-γ) secretion in response to several pools of cDNA clones. Further analysis of one particular pool revealed that only one of its constituents stimulated considerable IFN-γ secretion by CD4+T cells and that the expressed antigen is identical to a small fragment of myosin heavy chain.
A Localized Adherence-Like Pattern as a Second Pattern of Adherence of Classic Enteropathogenic
Escherichia coli
to HEp-2 Cells That Is Associated with Infantile Diarrhea
Authors: Scaletsky Isabel C. A. , Pedroso Margareth Z. , Oliva Carlos A. G. , Carvalho Rozane L. B. , Morais Mauro B. , Fagundes-Neto Ulysses
Published: December 2019

Escherichia coli
strains that cause nonbloody diarrhea in infants are known to present three distinct patterns of adherence to epithelial cells, namely, localized (LA), diffuse (DA), and aggregative (AA) adherence. Strains with LA (typical Enteropathogenic
Escherichia coli
[EPEC]) are well recognized as a cause of secretory diarrhea, but the role of strains with DA (DAEC) is controversial, and strains with AA (EAEC) have been more frequently related to persistent diarrhea whereas its relationship with acute diarrhea is not well defined. To determine the relationship of the different types of
E. coli
adherence patterns with acute diarrhea (lasting less than 14 days) and persistent diarrhea (lasting more than 14 days) in São Paulo, Brazil, we studied stool specimens from 40 infants under 1 year of age with diarrhea and 40 age-matched control infants without any gastrointestinal symptoms. Twenty-eight (35.0%) of eighty cases yielded adherent
E. coli
(HEp-2 cells). Strains with localized and aggregative adherence were associated with acute and persistent diarrhea. A total of 11.2% of the adherent strains were typical EPEC serotypes and hybridized with the enteroadherence factor probe; 5.0% were EAEC and hybridized with the EAEC probe. DAEC strains were isolated from 10.0% of patients and 7.5% of controls and did not hybridize with the two probes used (
daaC
and AIDA-I). Strains with a localized adherence-like pattern (atypical EPEC) were found significantly more frequently (
P
= 0.028) in cultures from children with diarrhea (17.5%) than in controls (2.5%).
Neisseria gonorrhoeae
Coordinately Uses Pili and Opa To Activate HEC-1-B Cell Microvilli, Which Causes Engulfment of the Gonococci
Authors: Griffiss J. McLeod , Lammel Claudia J. , Wang Jun , Dekker Nusi P. , Brooks G. F.
Published: December 2019

This study was undertaken to examine concomitant roles of pili and colony opacity-associated proteins (Opa) in promoting
Neisseria gonorrhoeae
adherence to and invasion of human endometrial HEC-1-B cells. Adherence of
N. gonorrhoeae
to cultured HEC-1-B cells was saturable, even though organisms adhered to <50% of the cells. During 4 to 6 h of incubation, adherent mono- and diplococci formed microcolonies on the surfaces of the cells. Microvilli of the HEC-1-B cells adhered by their distal ends to individual cocci within the microcolonies. When the microcolonies grew from isogenic pilus-negative (P
−
) Opa
−
, P
−
Opa
+
, or P
+
Opa
−
gonococci, microvilli did not elongate, and the colonies were not engulfed. In contrast, the microvilli markedly elongated during exposure to P
+
Opa
+
gonococci. The microvilli adhered to the organisms along their full lengths and appeared to actively participate in the engulfment of the microcolonies. Internalized microcolonies, with P
+
Opa
+
gonococci, contained dividing cocci and appeared to be surrounded by cell membrane but were not clearly within vacuoles. In contrast, degenerate individual organisms were within vacuoles. Low doses of chloramphenicol, which inhibits protein synthesis by both prokaryotes and eukaryotes, prevented the microvillar response to and internalization of the P
+
Opa
+
gonococci; higher doses caused internalization without microvillus activation. Cycloheximide and anisomycin, which inhibit only eukaryotic protein synthesis, caused dose-dependent enhancement of uptake. Cytochalasins reduced engulfment; colchicine had no effect. These results show that gonococci must express both pili and Opa to be engulfed efficiently by HEC-1-B cells.
Effect of
Escherichia coli
Cytotoxic Necrotizing Factor 1 on Repair of Human Bladder Cell Monolayers In Vitro
Authors: Island Michael D. , Cui Xaioling , Warren John W.
Published: December 2019

We hypothesized that
Escherichia coli
cytotoxic necrotizing factor 1 (CNF1) might impair migration or proliferation of bladder cells and could potentially interfere with repair of the bladder epithelium. Using experimentally wounded human T24 bladder epithelial cell monolayers as an in vitro model, we found that both the number of T24 cells and the maximum distance they migrated into wounded regions was significantly decreased by bacterial extracts containing
E. coli
CNF1.
Effect of Intrastrain Variation in the Amount of Capsular Polysaccharide on Genetic Transformation of
Streptococcus pneumoniae
: Implications for Virulence Studies of Encapsulated Strains
Authors: Weiser Jeffrey N. , Kapoor Miki
Published: December 2019

The degrees of competence of opaque and transparent colony variants of
Streptococcus pneumoniae
were compared. The transparent variants were transformed at 9- to 670-fold-higher rates than the opaque variants, independent of the DNA incorporated, due to decreased expression of capsular polysaccharide. Genetic transformation, therefore, tends to select for a less-encapsulated subpopulation.
Recombination betweengtfBandgtfCIs Required for Survival of a dTDP-Rhamnose Synthesis-Deficient Mutant ofStreptococcus mutansin the Presence of Sucrose
Authors: Yamashita Yoshihisa , Tomihisa Kiyotaka , Nakano Yoshio , Shimazaki Yoshihiro , Oho Takahiko , Koga Toshihiko
Published: December 2019

Thermlgenes are involved in dTDP-rhamnose synthesis inStreptococcus mutans. A gene fusion betweengtfBandgtfC, which both encode extracellular water-insoluble glucan-synthesizing enzymes, accompanied by inactivation of thermlgenes was observed for cells grown in the presence of sucrose. The survival rates ofrmlmutants isolated in the absence of sucrose were drastically reduced in the presence of sucrose. The rates were consistent with the frequency of spontaneous gene fusions betweengtfBandgtfC, suggesting that the spontaneous recombinant organisms were selected in the presence of sucrose. Thermlmutants with agtfB-gtfCfusion gene had markedly reduced water-insoluble glucan synthetic activity and lost the ability to colonize glass surfaces in the presence of sucrose. These results suggest that thermlmutants ofS. mutans, which are defective in dTDP-rhamnose synthesis, can survive only in the absence of water-insoluble glucan synthesis.
Vibrio cholerae
Intestinal Population Dynamics in the Suckling Mouse Model of Infection
Authors: Angelichio Michael J. , Spector Jonathon , Waldor Matthew K. , Camilli Andrew
Published: December 2019

The suckling mouse has been used as a model to identify
Vibrio cholerae
intestinal colonization factors for over two decades, yet little is known about the location of recoverable organisms along the gastrointestinal (GI) tract following intragastric inoculation. In the present study, we determined the population dynamics of wild-type and avirulent mutant derivatives of both classical and El Tor biotype strains throughout the entire suckling mouse GI tract at various times after intragastric inoculation. Wild-type strains preferentially colonized the middle small bowel with a sharp demarcation between more proximal segments which had manyfold-fewer recoverable cells. Surprisingly, large and stable populations of viable cells were also recovered from the cecum and large bowel. Strains lacking toxin-coregulated pili (TCP
−
) were cleared from the small bowel; however, an El Tor TCP
−
strain colonized the cecum and large bowel almost as well as the wild-type strain. Strains lacking lipopolysaccharide O antigen (OA
−
) were efficiently cleared from the small bowel at early times but then showed net growth for the remainder of the infections. Moreover, large populations of the OA
−
strains were maintained in the large bowel. These results show that for the El Tor biotype neither TCP nor OA is required for colonization of the suckling mouse large bowel. Finally, similar percent recoveries of wild-type, TCP
−
, and OA
−
strains from the small bowel at an early time after infection suggest that TCP and OA are not required for strains of either biotype to resist bactericidal mechanisms in the suckling mouse GI tract.
Target Cell Range of
Haemophilus ducreyi
Hemolysin and Its Involvement in Invasion of Human Epithelial Cells
Authors: Wood Gwendolyn E. , Dutro Susan M. , Totten Patricia A.
Published: December 2019

Haemophilus ducreyi
, the causative agent of chancroid, produces a hemolysin, whose role in virulence is not well defined. To assess the possible role of hemolysin in pathogenesis, we evaluated its target cell range by using wild-type
H. ducreyi
35000, nonhemolytic mutants with the hemolysin structural gene deleted, and isogenic strains expressing different amounts of hemolytic activity. The cytotoxicity of the various cell types was assessed by quantitating the release of lactate dehydrogenase into culture supernatants as a measure of cell lysis. In these experiments, human foreskin fibroblasts, human foreskin epithelial cells, and, to a lesser extent, HEp-2 cells were lysed by
H. ducreyi
hemolysin. Hemolysin also lysed human blood mononuclear cells and immune system cell lines including U937 macrophage-like cells, T lymphocytes, and B lymphocytes. In contrast, human polymorphonuclear leukocytes were not sensitive to hemolysin under the conditions tested. We also analyzed the effect of hemolysin on invasion of human epithelial cells and found that
H. ducreyi
strains expressing cloned hemolysin genes showed a 10-fold increase in invasion compared to the control strain. These data support the hypothesis that the
H. ducreyi
hemolysin is important in the pathogenesis of chancroid and may contribute to ulcer formation, invasion of epithelial cells, and evasion of the immune response.
Predominance of CD4 Th1 and CD8 Tc1 Cells Revealed by Characterization of the Cellular Immune Response Generated by Immunization with a DNA Vaccine Containing aTrypanosoma cruziGene
Authors: Rodrigues Mauricio M. , Ribeirão Marcelo , Pereira-Chioccola Vera , Renia Laurent , Costa Fabio
Published: December 2019

Immunization with a plasmid DNA containing the gene encoding the catalytic domain oftrans-sialidase (TS) elicits protective immune responses against experimentalTrypanosoma cruziinfection. As several studies provided strong evidence that during infection CD4 Th1 and CD8 T cytotoxic type 1 (Tc1) cells are important factors in host resistance, the present study was designed to evaluate which T-cell types were activated in DNA-vaccinated BALB/c mice. We found that bulk cells from DNA-immunized mice had CD4 and CD8 T cells that produced gamma interferon (IFN-γ) but not interleukin-4 (IL-4) or IL-10. To characterize the TS-specific T cells at the clonal level, we generated CD4 and CD8 clones. We obtained cytotoxic CD4 clones of the Th1 type that secreted large amounts of IFN-γ but not IL-4 or IL-10. Unexpectedly, we obtained other CD4 clones with a Th2 phenotype, secreting IL-4 and IL-10 but not IFN-γ. All CD8 clones were cytotoxic and produced IFN-γ. IL-4 and IL-10 were not secreted by these cells. Using synthetic peptides, we determined a CD8 epitope recognized by several clones as being represented by amino acids IYNVGQVSI. The antiparasitic activity of a CD4 Th1 and a CD8 Tc1 clone was assessed in vitro. CD4 or CD8 T cells significantly inhibitedT. cruzidevelopment in infected macrophages or fibroblasts, respectively. We concluded that DNA vaccine efficiently generates potentially protective CD4 Th1 and CD8 Tc1 cells specific for aT. cruziantigen, therefore reinforcing the possibility of using this strategy for developing a preventive or therapeutic vaccine against Chagas’ disease.
Essential Role of Transcription Factor Nuclear Factor-κB in Regulation of Interleukin-8 Gene Expression by Nitrite Reductase fromPseudomonas aeruginosain Respiratory Epithelial Cells
Authors: Mori Naoki , Oishi Kazunori , Sar Borann , Mukaida Naofumi , Nagatake Tsuyoshi , Matsushima Kouji , Yamamoto Naoki
Published: December 2019

Persistent infection withPseudomonas aeruginosaincreases interleukin-8 (IL-8) levels and causes dense neutrophil infiltrations in the airways of patients with chronic airway diseases. Recently, we have reported that nitrite reductase fromP. aeruginosainduces the production of IL-8 in respiratory cells, including bronchial epithelial cells. To determine the molecular mechanism(s) of nitrite reductase-induced IL-8 expression in respiratory cells, A549 epithelial cells were transfected with plasmids containing serial deletions of the 5′-flanking region of the IL-8 gene and then exposed to nitrite reductase. Nitrite reductase significantly enhanced IL-8 gene promoter-driven reporter activity. This increased IL-8 gene expression was inhibited by mutating the nuclear factor-κB (NF-κB) binding element. Nitrite reductase enhanced nuclear localization of the NF-κB binding complex. Furthermore, nitrite reductase induced the degradation of IκBα, the major cytoplasmic inhibitor of NF-κB, and the expression of IκBα mRNA. These data support the critical role of the activation of NF-κB in nitrite reductase-induced IL-8 gene expression in airway epithelium.
Characterization of a
Haemophilus ducreyi
Mutant Deficient in Expression of Cytolethal Distending Toxin
Authors: Stevens Marla K. , Latimer Jo L. , Lumbley Sheryl R. , Ward Christine K. , Cope Leslie D. , Lagergard Teresa , Hansen Eric J.
Published: December 2019

Haemophilus ducreyi
expresses a soluble cytolethal distending toxin (CDT) that kills HeLa, HEp-2, and other human epithelial cells in vitro.
H. ducreyi
CDT activity is encoded by a three-gene cluster (
cdtABC
), and antibody to the
cdtC
gene product can neutralize CDT activity in vitro (L. D. Cope, S. R. Lumbley, J. L. Latimer, J. Klesney-Tait, M. K. Stevens, L. S. Johnson, M. Purven, R. S. Munson, Jr., T. Lagergard, J. D. Radolf, and E. J. Hansen, Proc. Natl. Acad. Sci. USA 94:4056–4061, 1997). Culture supernatant fluid from a recombinant
Escherichia coli
strain containing the
H. ducreyi cdtABC
gene cluster readily killed both HeLa cells and HaCaT keratinocytes and had a modest inhibitory effect on the growth of human foreskin fibroblasts. Insertional inactivation of the
cdtC
gene in this recombinant
E. coli
strain eliminated the ability of this strain to kill HeLa cells and HaCaT keratinocytes. This mutated
H. ducreyi cdtABC
gene cluster was used to construct an isogenic
H. ducreyi cdtC
mutant. Monoclonal antibodies against the
H. ducreyi
CdtA, CdtB, and CdtC proteins were used to characterize protein expression by this
cdtC
mutant. Culture supernatant fluid from this
H. ducreyi cdtC
mutant did not detectably affect any of the human cells used in this study. The presence of the wild-type
H. ducreyi cdtC
gene in
trans
in this
H. ducreyi
mutant restored its ability to express a CDT that killed both HeLa cells and HaCaT keratinocytes. The isogenic
H. ducreyi cdtC
mutant was shown to be as virulent as its wild-type parent strain in the temperature-dependent rabbit model for experimental chancroid. Lack of expression of the
H. ducreyi
CdtC protein also did not affect the ability of this
H. ducreyi
mutant to survive in the skin of rabbits.
Early Emergence of CD8+T Cells Primed for Production of Type 1 Cytokines in the Lungs ofMycobacterium tuberculosis-Infected Mice
Authors: Serbina Natalya V. , Flynn JoAnne L.
Published: December 2019

Several lines of evidence suggest that CD8 T cells are important in protection against tuberculosis. To understand the function of this cell population in the immune response againstMycobacterium tuberculosis, T cells from lungs ofM. tuberculosis-infected mice were examined by flow cytometry. The kinetics of the appearance of CD8 T cells in lungs of infected mice closely paralleled that of CD4 T cells. Both CD4+and CD8+T cells displaying an activated phenotype were found in the lungs as early as 1 week postinfection. By 2 weeks, total cell numbers in the lungs had tripled and percentages of T cells were increased two- to threefold; the percentages of CD4+T cells were ca. twofold higher than those of CD8+T cells. Short-term stimulation withM. tuberculosis-infected antigen-presenting cells induced cytokine production by primed CD4+and CD8+T cells. Intracellular cytokine staining revealed that 30% ± 5% of CD4+and 23% ± 4% of CD8+T cells were primed for production of gamma interferon (IFN-γ). However, a difference in in vivo IFN-γ production by T cells was observed with ∼12% of CD4+T cells and ∼5% of CD8+T cells secreting cytokine in the lungs at any given time during infection. The data presented indicate that although early in infection the majority of IFN-γ is produced by CD4+T cells, cytokine-producing CD8+T cells are readily available when triggered by the appropriate stimuli.
Interaction ofBrucella abortusLipopolysaccharide with Major Histocompatibility Complex Class II Molecules in B Lymphocytes
Authors: Forestier Claire , Moreno Edgardo , Méresse Stéphane , Phalipon Armelle , Olive Daniel , Sansonetti Philippe , Gorvel Jean-Pierre
Published: December 2019

Lipopolysaccharide (LPS), a major amphiphilic molecule located at the outer membrane of gram-negative bacteria, is a potent antigen known to induce specific humoral immune responses in infected mammals. LPS has been described as a polyclonal activator of B lymphocytes, triggering the secretion of antibodies directed against distinct sugar epitopes of the LPS chain. But, how LPS is handled by B cells remains to be fully understood. This task appears to be essential for a better knowledge of the anti-LPS humoral immune response. In this study, we examine the internalization of LPS and its interaction with antigen-presenting major histocompatibility complex (MHC) class II molecules in murine and human B-cell lines. By use of immunofluorescence, we observe that structurally different LPSs fromBrucellaandShigellastrains accumulate in an intracellular compartment enriched in MHC class II molecules. By use of immunoprecipitation, we illustrate that onlyBrucella abortusLPS associates with MHC class II molecules in a haplotype-independent manner. Taken together, these results raise the possibility thatB. abortusLPS may play a role in T-cell activation.
Opacity-Associated Protein A Contributes to the Binding of
Haemophilus influenzae
to Chang Epithelial Cells
Authors: Prasadarao Nemani V. , Lysenko Elena , Wass Carol A. , Kim Kwang Sik , Weiser Jeffrey N.
Published: December 2019

Opacity-associated protein A (OapA), which is responsible for the transparent-colony phenotype of
Haemophilus influenzae
, has been implicated in the colonization of the nasopharynx in an infant rat model of carriage. In this report, we show that OapA mediates attachment to Chang epithelial cells examined by using genetically defined type b and nontypeable
H. influenzae
strains with or without OapA. We also showed that OapA was conserved among
H. influenzae
strains by comparing deduced amino acid sequences. Both recombinant OapA and polyclonal anti-OapA antiserum blocked the binding of
H. influenzae
to Chang epithelial cells, suggesting that the interaction of
H. influenzae
is specific to OapA. Moreover, the binding of recombinant OapA to epithelial cells further provided evidence that OapA can promote attachment of
H. influenzae
. Expression of
oapA
gene in a nonadherent
Escherichia coli
strain significantly increased the binding to Chang epithelial cells, and disruption of the
oapA
gene with kanamycin resistance cassette insertion resulted in a significant loss of binding. These findings demonstrate that OapA plays a role in
H. influenzae
binding to human conjunctival epithelial cells.
Campylobacter jejuni
81-176 Associates with Microtubules and Dynein during Invasion of Human Intestinal Cells
Authors: Hu Lan , Kopecko Dennis J.
Published: December 2019

Campylobacter jejuni
uptake into cultured INT407 cells was analyzed kinetically over a wide range of starting multiplicities of infection (MOI; from 0.02 to 20,000 bacteria/epithelial cell). The efficiency of internalization was the highest at MOI of 0.02 and decreased steadily at higher MOIs, presumably due to reported
C. jejuni
autoagglutination at higher densities. Total internalized
Campylobacter
CFU increased gradually from an MOI of 0.02 to a peak at an MOI of 200 (reaching an average of two bacteria internalized per epithelial cell) and decreased at higher MOIs. The invasion process was apparently saturated within 2 h at an MOI of 200, indicating stringent host cell limitations on this entry process. Furthermore, whereas control
Salmonella typhi
invaded all monolayer cells within 1 h, only two-thirds of monolayer cells were infected after 2 h with
C. jejuni
at MOIs of 200 to 2,000. The percentage of
Campylobacter
-infected host cells gradually increased to 85% after 7 h of infection, suggesting that
C. jejuni
entry may be host cell cycle dependent. Direct evidence of the involvement of microtubules in
C. jejuni
internalization, suggested previously by biochemical inhibitor studies, was obtained by time course immunofluorescence microscopic analyses. Bacteria initially bound to the tips of host cell membrane extensions containing microtubules, then aligned in parallel with microtubules during entry, colocalized specifically with microtubules and dynein but not with microfilaments, and moved over 4 h, presumably via microtubules to the perinuclear region of host cells. Orthovanadate, which inhibits dynein activity, specifically reduced
C. jejuni
81-176 entry, suggesting that this molecular motor is involved in entry and endosome trafficking during this novel bacterial internalization process. Collectively, these data suggest that
C. jejuni
enters host cells in a targeted and tightly controlled process leading to uptake into an endosomal vacuole which apparently moves intracellularly along microtubules via the molecular motor, dynein, to the perinuclear region.
Plasma Membrane Expression of Heat Shock Protein 60 In Vivo in Response to Infection
Authors: Belles Cindy , Kuhl Alicia , Nosheny Rachel , Carding Simon R.
Published: December 2019

Heat shock protein 60 (hsp60) is constitutively expressed in the mitochondria of eukaryotic cells. However, it has been identified in other subcellular compartments in several disease states and in transformed cells, and it is an immunogenic molecule in various infectious and autoimmune diseases. To better understand the factors that influence expression of hsp60 in normal cells in vivo, we analyzed its cellular and subcellular distribution in mice infected with the intracellular bacteriumListeria monocytogenes. Western blotting of subcellular fractionated spleen cells showed that although endogenous hsp60 was restricted to the mitochondria in noninfected animals, it was associated with the plasma membrane as a result of infection. The low levels of plasma membrane-associated hsp60 seen in the livers in noninfected animals subsequently increased during infection. Plasma membrane hsp60 expression did not correlate with bacterial growth, being most evident during or after bacterial clearance and persisting at 3 weeks postinfection. Using flow cytometry, we determined that Mac-1+, T-cell receptor γδ+, and B220+cells represented the major Hsp60+populations in spleens of infected mice. By contrast, B220+cells were the predominant hsp60+population in livers of infected mice. Of the immune cells analyzed, the kinetic profile of the γδ T-cell response most closely matched that of hsp60 expression in both the spleen and liver. Collectively, these findings show that during infection hsp60 can be localized to the plasma membrane of viable cells, particularly antigen-presenting cells, providing a means by which hsp60-reactive lymphocytes seen in various infectious disease and autoimmune disorders may be generated and maintained.
Intranasal Immunization of Mice with Influenza Vaccine in Combination with the Adjuvant LT-R72 Induces Potent Mucosal and Serum Immunity Which Is Stronger than That with Traditional Intramuscular Immunization
Authors: Barackman J. D. , Ott G. , O’Hagan D. T.
Published: December 2019

Immunization of mice by the intranasal route with influenza virus hemagglutinin in combination with the mutantEscherichia coliheat-labile enterotoxin R72 (LT-R72) induced significantly enhanced serum and mucosal antibodies, surpassing, in most cases, responses achieved by traditional intramuscular immunization using inactivated split influenza vaccine. Furthermore, intranasal immunization with LT-R72 induced a potent serum immunoglobulin G2a response, indicating that this adjuvant has Th1 character.