bluuebunny/crossref_metadata_2025 huggingface dataset:
Genes Influencing Resistance toCoccidioides immitisand the Interleukin-10 Response Map to Chromosomes 4 and 6 in Mice
Authors: Fierer Joshua , Walls Lorraine , Wright Fred , Kirkland Theo N.
Published: December 2019

Coccidioidomycosis is a fungal infection that is endemic in the southwestern United States. Infection is more severe in blacks and Filipinos, which suggests that there is a genetic basis for susceptibility to this infection in humans. We found that there is also a difference in resistance toCoccidioides immitisinfection among inbred mouse strains: B6 mice are susceptible, while DBA/2 mice are resistant (T. N. Kirkland and J. Fierer, Infect. Immun. 40:912–916, 1983). In this paper we report the results of our efforts to map the genes responsible for resistance to this infection in mice. Mice were infected by intraperitoneal inoculation, and 15 days later the numbers of viable fungi in their lungs and spleens were enumerated. We also determined the amounts of interleukin-10 mRNA made in the infected lungs. These three phenotypes were mapped as quantitative traits by using the 26 available lines of recombinant inbred mice derived from a cross between B6 and DBA/2 mice. The best associations were those between the regions near theLvlocus on chromosome 4 and theTnfr1locus on chromosome 6. We then infected backcross mice [(B6 × DBA/2) × B6] and confirmed these associations; 14 of 16 (87%) mice that were heterozygous at bothLvandTnfr1were resistant to infection, whereas only 4 of 16 (25%) mice that were homozygous B6 at both loci were resistant. These are the first genetic loci to be associated with susceptibility toC. immitis, but there may be additional genes involved in murine resistance to this infection.
Disruption of Anthrax Toxin Binding with the Use of Human Antibodies and Competitive Inhibitors
Authors: Cirino Nick M. , Sblattero Daniele , Allen David , Peterson Scott R. , Marks James D. , Jackson Paul J. , Bradbury Andrew , Lehnert Bruce E.
Published: December 2019

The protective antigen (PA83) of
Bacillus anthracis
is integral to the mechanism of anthrax toxicity. We have isolated a human single-chain Fv antibody fragment (scFv) that blocks binding of a fluorescently tagged protective antigen (PA) moiety to cell surface receptors. Several phage-displayed scFv were isolated from a naive library biopanned against PA83. Soluble, monomeric scFv were characterized for affinity and screened for their capacity to disrupt receptor-mediated binding of PA. Four unique scFv bound to PA83, as determined by surface plasmon resonance, the tightest binder exhibiting a

K
d

of 50 nM. Two scFv had similar affinities for natural PA83 and a novel, recombinant, 32-kDa carboxy-terminal PA fragment (PA32). Binding of scFv to green fluorescent protein fused to the amino-terminal 32-kDa fragment of
B. anthracis
edema factor, EGFP-EF32, was used to confirm specificity. Fusion of EGFP to PA32 facilitated development of a novel flow cytometric assay that showed that one of the scFv disrupted PA receptor binding. This method can now be used as a rapid assay for small molecule inhibitors of PA binding to cell receptors. The combined data presented suggest the potential utility of human scFv as prophylactics against anthrax poisoning. Moreover, recombinant PA32 may also be useful as a therapeutic agent to compete with anthrax toxins for cellular receptors during active infection.
Modulation of Major Histocompatibility Complex Protein Expression by Human Gamma Interferon Mediated by Cysteine Proteinase-Adhesin Polyproteins ofPorphyromonas gingivalis
Authors: Yun Peter L. W. , DeCarlo Arthur A. , Hunter Neil
Published: December 2019

Cysteine proteinases have been emphasized in the virulence ofPorphyromonas gingivalisin chronic periodontitis. These hydrolases may promote the degradation of extracellular matrix proteins and disrupt components of the immune system. In this study it was shown that purified Arg-gingipain and Lys-gingipain inhibited expression of class II major histocompatibility complex (MHC) proteins in response to the stimulation of endothelial cells with human gamma interferon (IFN-γ). Treatment with the cysteine proteinases resulted in a rapid shift in the apparent molecular size of IFN-γ from 17 to 15 kDa, as shown by Western blot analysis, a response which also occurred in the presence of serum. Further, glycosylated natural IFN-γ from human leukocytes and unglycosylated recombinant IFN-γ fromEscherichia coliwere both digested by the cysteine proteinases. Immunoblot analysis indicated that cleavage within the carboxyl terminus of recombinant IFN-γ correlated with the loss of induction of MHC class II expression as monitored by analytical flow cytometry. No hydrolysis of MHC class II molecules or human IFN-γ receptor by these proteinases was detected by Western blot analysis. These findings suggest thatP. gingivaliscysteine proteinases may alter the cytokine network at the point of infection through the cleavage of IFN-γ. Degradation of IFN-γ could have important consequences for the recruitment and activation of leukocytes and therefore may contribute significantly to the destruction of the periodontal attachment.
Citrobacter rodentiumInfection in Mice Elicits a Mucosal Th1 Cytokine Response and Lesions Similar to Those in Murine Inflammatory Bowel Disease
Authors: Higgins Lisa M. , Frankel Gad , Douce Gill , Dougan Gordon , MacDonald Thomas T.
Published: December 2019

Citrobacter rodentiumis a classically noninvasive pathogen of mice that is similar to enteropathogenicEscherichia coli(EPEC) in man. Following oral infection of young mice, the organism colonizes the distal colon, and within 1 week the colonic mucosa doubles in thickness and there is massive epithelial cell hyperplasia. Since T-cell responses in mouse models of inflammatory bowel disease (IBD) also cause epithelial hyperplasia, we have investigated the possibility thatC. rodentiumpromotes similar T-cell responses in the mucosa, thereby increasing epithelial shedding, transmission, and replication of the organism. Beginning 6 days after infection, bacteria were observed to be in close association with the epithelial surface and were also visible scattered throughout the lamina propria and in the submucosa. There was a CD3+-cell infiltrate into the colonic lamina propria and epithelium as well as mucosal thickening and crypt hyperplasia. The majority of CD3+cells were CD4+and were not γδ+. Reverse transcription-PCR analysis of cytokines also revealed a highly polarized Th1 response (interleukin-12, gamma interferon, and tumor necrosis factor alpha) in the mucosa and a large increase in the epithelial cell mitogen keratinocyte growth factor. None of the changes were seen in mice inoculated with bacteria lacking intimin (which is necessary for colonization), but they were seen in mice inoculated withC. rodentiumcomplemented with intimin from EPEC. This is the first example of a classically noninvasive bacterial pathogen which elicits a strong mucosal Th1 response and which produces pathology similar to that seen in mouse models of IBD, which is also characterized by a strong Th1 response. These results also suggest that the colonic mucosa responds in a stereotypic way to Th1 responses.
Coinfection with Influenza B Virus Does Not Affect Association ofNeisseria meningitidiswith Human Nasopharyngeal Mucosa in Organ Culture
Authors: Read Robert C. , Goodwin Linda , Parsons M. Andrew , Silcocks Paul , Kaczmarski Edward B. , Parker Andrew , Baldwin Thomas J.
Published: December 2019

There is an epidemiological association between influenza virus infection and meningococcal disease. Proposed mechanisms are the destruction of the normal epithelial barrier function of the upper respiratory tract by influenza virus or the expression of human or viral surface-exposed proteins that enhance bacterial adherence and/or invasion. To test these hypotheses, human nasopharyngeal mucosa specimens from a total of 19 individual donors were successfully infected with influenza B virus and then inoculated with serogroup BNeisseria meningitidis. Subsequent bacterial association with the epithelial surface was measured in three separate series of experiments by using transmission electron microscopy (n= 6), scanning electron microscopy (n= 6), and counting of viable bacteria within homogenates of explants (n= 7). Penetration of the mucosa was estimated by measuring the count of viable bacteria recovered from explants after exposure to sodium taurocholate. Bacterial association with the surface of explants was time dependent over 24 h of superinfection. Influenza virus did not positively or negatively influence bacterial attachment to or penetration of explant mucosa compared to those of uninfected controls, even when the period of preincubation with virus was extended to 7 days. When proteins were purified from mucosal epithelium and immobilized on nitrocellulose membranes,N. meningitidisattached predominantly to bands corresponding to proteins of 210 and 130 kDa. In the presence of influenza virus infection, these proteins were gradually lost over the course of 72 h. In conclusion, influenza B virus did not increase association of serogroup BN. meningitidiswith human nasopharyngeal mucosa.
Increased Antimycobacterial Immunity in Interleukin-10-Deficient Mice
Authors: Murray Peter J. , Young Richard A.
Published: December 2019

Macrophage effector functions are essential for clearing mycobacterial infections. Interleukin 10 (IL-10) negatively regulates macrophages and could be a factor inhibiting effective antimycobacterial immunity. We previously showed that transgenic mice which produce excess IL-10 from T cells are susceptible to infection, even though these mice continue to produce gamma interferon (IFN-γ) at levels similar to those in controls. Here, we extend our genetic analysis of the functions of IL-10 in antimycobacterial immunity by testing the hypothesis that IL-10-deficient (IL-10−/−) mice should be more resistant to mycobacteria than control mice.Mycobacterium bovisbacillus Calmette-Guérin-infected IL-10−/−mice had significantly lower bacterial burdens than control mice early in the infection. Contrary to expectations, however, IL-10−/−mice did not have increased levels of IFN-γ, either from T cells or in the plasma, suggesting that other mechanisms are responsible for the increased resistance. However, macrophages from IL-10−/−mice produced increased levels of inflammatory cytokines, including IFN-γ, as well as nitric oxide and prostaglandins, which could account for increased antimycobacterial immunity. Our genetic analysis revealed that IL-10 is an inhibitor of early mycobacterial clearance. The data also suggest that IL-10 negatively regulates numerous macrophage functions as well as playing a role in down-regulating the general inflammatory response, especially in conditions where an infection must be controlled through macrophage activity.
In Vitro and In Vivo Stability aCryptococcus neoformansGlucuronoxylomannan Epitope That Elicits Protective Antibodies
Authors: Cleare Wendy , Cherniak Robert , Casadevall Arturo
Published: December 2019

The monoclonal antibody (MAb) 2H1 defines an epitope inCryptococcus neoformanscapsular glucuronoxylomannan (GXM) that can elicit protective antibodies. In murine models of cryptococcosis, MAb 2H1 administration prolongs survival and reduces fungal burden but seldom clears the infection. The mechanism by whichC. neoformanspersists and escape antibody-mediated clearance is not understood. One possibility is that variants that do not bind MAb 2H1 emerge in the course of infection. Using an agglutination-sedimentation protocol, we recovered a variant of strain 24067 that did not agglutinate, could not be serotyped, and had marked reduction in GXMO-acetyl groups. Binding of MAb 2H1 to 24067 variant cells produced a different immunofluorescence pattern and lower fluorescence intensity relative to the parent 24067 cells. Addition of MAb 2H1 to 24067 variant cells had no effect on cell charge. Phagocytic assays demonstrated that MAb 2H1 was not an effective opsonin for the 24067 variant. The 24067 variant was less virulent than the 24067 parent strain in mice, and MAb 2H1 administration did not prolong survival in animals infected with the variant strain. To investigate whether variants which do not bind MAb 2H1 are selected in experimental infection, threeC. neoformansstrains were serially passaged in mice given either MAb 2H1 or no antibody. Analysis of passaged isolates by agglutination assay, flow cytometry, and indirect immunofluorescence revealed changes in MAb 2H1 epitope expression but no clear trend with regards to gain or loss of MAb 2H1 epitope.C. neoformansvariants with reduced MAb 2H1 epitope content can be isolated in vitro, but persistence of infection in mice given MAb 2H1 does not appear to be a result of selection of escape variants that lack the MAb 2H1 epitope.
Analysis of Vaginal Cell Populations during Experimental Vaginal Candidiasis
Authors: Fidel Paul L. , Luo Wei , Steele Chad , Chabain Joseph , Baker Marc , Wormley Floyd
Published: December 2019

Studies with an estrogen-dependent murine model of vaginal candidiasis suggest that local cell-mediated immunity (CMI) is more important than systemic CMI for protection against vaginitis. The present study, however, showed that, compared to uninfected mice, little to no change in the percentage or types of vaginal T cells occurred during a primary vaginal infection or during a secondary vaginal infection where partial protection was observed. Furthermore, depletion of polymorphonuclear leukocytes (PMN) had no effect on infection in the presence or absence of pseudoestrus. These results indicate a lack of demonstrable effects by systemic CMI or PMN against vaginitis and suggest that if local T cells are important, they are functioning without showing significant increases in numbers within the vaginal mucosa during infection.
Promoter Architecture of the
Porphyromonas gingivalis
Fimbrillin Gene
Authors: Xie Hua , Lamont Richard J.
Published: December 2019

Porphyromonas gingivalis
fimbriae can mediate adherence to many of the available substrates in the oral cavity. Expression of
P. gingivalis
fimbriae is regulated at the transcriptional level by environmental signals, such as temperature and hemin concentration. The arrangement of the upstream promoter and regulatory sequences required for transcription and control of the fimbrial structural gene (
fimA
) was investigated. Primer extension analysis demonstrated that the transcriptional start site of the
fimA
gene is located 41 bp upstream from the translational start codon. A region (
upf
) spanning 648 bp upstream of the start codon to 44 bp downstream of the translational start site was cloned upstream of a promoterless
lacZ
reporter gene. A series of deletion and base substitution mutations were then generated in the
upf
region. The constructs were introduced into the chromosome of
P. gingivalis
, and promoter activity measured by assaying levels of β-galactosidase. The results showed that
fimA
contains sequences resembling ς
70
promoter consensus sequences, consisting of a −10 region (TATGAC) located at −18 to −23 and a −35 region (TTGTTG) located at −41 to −46 from the transcriptional start point. The AT-rich upstream sequences spanning bases −48 to −85 and bases −90 to −240 were required for full expression of the
fimA
gene, indicating the existence of positive regulation regions. Moreover, the −48 to −64 region may constitute an UP element, contributing to promoter activity in
P. gingivalis
. Thus, our data suggest that the
P. gingivalis fimA
gene has a transcription complex consisting of −10 and −35 sequences, an UP element, and additional AT-rich upstream regulatory sequences.
Coxiella burnetiiInfection Increases Transferrin Receptors on J774A.1 Cells
Authors: Howe Dale , Mallavia Louis P.
Published: December 2019

Inoculation with viable, but not inactivated,Coxiella burnetiiresulted in the increased expression of transferrin receptors (TfR) in the murine macrophage-like cell line J774A.1. This upregulation was evident in immunoblots as early as 6 h postinfection, with TfR levels continuing to increase through the first 24 h of infection. Fluorescent labeling revealed that TfR upregulation occurred throughout infected monolayers, eliminating the possibility that it reflected a response by a minor subset of host cells. In addition, TfR trafficking did not appear to be affected byC. burnetiiinfection. Consistent with the increase in TfRs, inoculation with viableC. burnetiiresulted in a 2.5-fold increase in total cellular iron by 12 h postinoculation. Our further findings that the chelation of intracellular iron arrestsC. burnetiireplication and thatC. burnetiimetabolic activities in vitro are affected by iron concentration suggest that TfR upregulation is a salient factor inC. burnetiiinfection, and we speculate that it may represent a significant virulence mechanism.
Cytotoxic T-Lymphocyte Epitopes Fused to Anthrax Toxin Induce Protective Antiviral Immunity
Authors: Doling Amy M. , Ballard Jimmy D. , Shen Hao , Krishna Kaja Murali , Ahmed Rafi , Collier R. John , Starnbach Michael N.
Published: December 2019

We have investigated the use of the protective antigen (PA) and lethal factor (LF) components of anthrax toxin as a system for in vivo delivery of cytotoxic T-lymphocyte (CTL) epitopes. During intoxication, PA directs the translocation of LF into the cytoplasm of mammalian cells. Here we demonstrate that antiviral immunity can be induced in BALB/c mice immunized with PA plus a fusion protein containing the N-terminal 255 amino acids of LF (LFn) and an epitope from the nucleoprotein (NP) of lymphocytic choriomeningitis virus. We also demonstrate that BALB/c mice immunized with a single LFn fusion protein containing NP and listeriolysin O protein epitopes in tandem mount a CTL response against both pathogens. Furthermore, we show that NP-specific CTL are primed in both BALB/c and C57BL/6 mice when the mice are immunized with a single fusion containing two epitopes, one presented by Ldand one presented by Db. The data presented here demonstrate the versatility of the anthrax toxin delivery system and indicate that this system may be used as a general approach to vaccinate outbred populations against a variety of pathogens.
Prevalence of, Antibody Response to, and Immunity Induced byHaemophilus ducreyiHemolysin
Authors: Dutro Susan M. , Wood Gwendolyn E. , Totten Patricia A.
Published: December 2019

Haemophilus ducreyi, the etiologic agent of chancroid, a genital ulcer disease, produces a cell-associated hemolysin whose role in virulence is not well defined. Hemolysin is encoded by two genes,hhdAandhhdB, which, based on their homology toSerratia marcescens shlAandshlBgenes, are believed to encode the hemolysin structural protein and a protein required for secretion and modification of this protein, respectively. In this study, we determined the prevalence and expression of the hemolysin genes in 90H. ducreyiisolates obtained from diverse geographic locations from 1952 to 1996 and found that all strains contained DNA homologous to thehhdBandhhdAgenes. In addition, all strains expressed a hemolytic activity. We also determined that hemolysin is expressed in vivo and is immunogenic, as indicated by the induction of antibodies to hemolysin in both the primate and rabbit disease models as well as in human patients with naturally acquired chancroid. Wild-type strain 35000 and isogenic hemolysin-negative mutants showed no difference in lesion development in the temperature-dependent rabbit model. However, immunization of rabbits with the purified hemolysin protein reduced the recovery of wild-typeH. ducreyi, but not hemolysin-negative mutants, from lesions. Our study indicates that hemolysin is a possible candidate for vaccine development due to its immunogenicity, expression in vitro and in vivo by most, if not all, strains, and the effect of immunization on reducing the recovery of viableH. ducreyiin experimental disease in rabbits.
Schistosoma mansoniActivates Host Microvascular Endothelial Cells To Acquire an Anti-Inflammatory Phenotype
Authors: Trottein Francois , Descamps Laurence , Nutten Sophie , Dehouck Marie-Pierre , Angeli Veronique , Capron Andre , Cecchelli Romeo , Capron Monique
Published: December 2019

Since endothelial cells (ECs) play a key role in immune defense mechanisms and in immunopathology, we investigated whether the intravascular helminth parasiteSchistosoma mansonicould interact with and activate resting ECs in vitro. Microscopic analysis revealed that the lung-stage schistosomula specifically attached to microvascular ECs. This adherence was associated to active cellular processes involving actin filament formation. Since variation of permeability of cultured capillary brain ECs is a good marker for endothelial activation, the transendothelial passage of a low-molecular-weight molecule (inulin) on monolayers of bovine brain capillary ECs (BBCEC) was measured in response to parasites. Schistosomula induced a dramatic decrease in transendothelial permeability, a characteristic marker for the generation of an anti-inflammatory phenotype to ECs. This paracellular barrier enhancing effect on endothelial monolayers was due to a soluble substance(s) (below 1 kDa in size) secreted fromS. mansonischistosomula and not by mechanisms associated to adherence between parasites and ECs. The reinforcement of the endothelial barrier function was accompanied by an elevation of intracellular concentration of cyclic AMP (cAMP). The use of specific kinase inhibitors confirms that schistosomula activate ECs through a cAMP/protein kinase A pathway that leads to an increased phosphorylation of the myosin light-chain kinase. These combined findings suggest that the secretory/excretory products from schistosomula possess anti-inflammatory factor(s) that signal host microvascular endothelium. The immunological consequences of such activation are discussed.
Chemokine Gene Expression duringPneumocystis carinii-Driven Pulmonary Inflammation
Authors: Wright Terry W. , Johnston Carl J. , Harmsen Allen G. , Finkelstein Jacob N.
Published: December 2019

Severe combined immunodeficient (SCID) mice lack functional lymphocytes and therefore developPneumocystis cariniipneumonia. However, when infected SCID mice are immunologically reconstituted with congenic spleen cells, a protective inflammatory cascade is initiated. Proinflammatory cytokines are produced, and lymphocytes and macrophages are recruited specifically to alveolar sites of infection. Importantly, uninfected regions of the lung remain free from inflammatory involvement, suggesting that there are specific mechanisms that limit inflammation in the infected lung. Therefore, to determine whether chemokines are involved in targeting theP. carinii-driven inflammatory response, steady-state mRNA levels of several chemokines were measured in the lungs of both reconstituted and nonreconstitutedP. carinii-infected SCID mice. Despite significant organism burdens in the lungs of 8- and 10-week-old SCID mice, there was no evidence of elevated chemokine gene expression, which is consistent with the lack of an inflammatory response in these animals. However, when 8-week-old infected SCID mice were immunologically reconstituted, signs of focal pulmonary inflammation were observed, and levels of RANTES, MCP-1, lymphotactin, MIP-1α, MIP-1β, and MIP-2 mRNAs were all significantly elevated. Chemokine mRNA abundance was elevated at day 10 postreconstitution (PR), was maximal at day 12 PR, and returned to baseline by day 22 PR. In situ hybridization demonstrated that during the peak of inflammation, RANTES gene expression was localized to sites of inflammatory cell infiltration andP. cariniiinfection. Thus, these observations indicate that chemokines play a role in the focal targeting of inflammatory cell recruitment to sites ofP. cariniiinfection after the passive transfer of lymphocytes to the host.
Dissemination ofListeria monocytogenesby Infected Phagocytes
Authors: Drevets Douglas A.
Published: December 2019

In vitro data suggest that blood-borneListeria monocytogenesorganisms enter the central nervous system (CNS) by direct invasion of endothelial cells or by cell-to-cell spread from infected phagocytes to endothelial cells. However, a role for infected phagocytes in neuroinvasion and dissemination ofL. monocytogenesin vivo has not been confirmed experimentally. Experiments described here tested whetherL. monocytogenes-infected peripheral blood leukocytes (PBL) circulated in bacteremic mice and could establish organ infection in vivo. A mean of 30.5% of bacteria cultured from whole blood were PBL associated, and microscopy showed that 22.2% of monocytes and 1.6% of neutrophils were infected. PBL-associated bacteria spread to endothelial cells in vitro, indicating their potential for virulence in vivo. To test this possibility, mice were injected intravenously with infected PBL and CFU of bacteria in liver, spleen, and brain were quantified and compared with values for mice injected with broth-grown bacteria and in vitro-infected macrophage cell lines. An inoculum of infected macrophage cell lines led to greater numbers of bacteria in the liver than the numbers produced by a similar inoculum of broth-grown bacteria. In contrast, brain infection was best established by infected PBL. Results of intraperitoneal injection of infected peritoneal cells compared with results of injection with infected J774A.1 cells suggested that unrestricted intracellular bacterial replication within J774A.1 cells contributed to excessive liver infection in those mice. These data show dissemination of intracellularL. monocytogenesand indicate that phagocyte-facilitated invasion has a role in CNS infection in vivo. Heterogeneity with regard to bactericidal activity as well as to other phagocyte characteristics is a critical feature of this mechanism.
Role of the 85-Kilobase Plasmid and Plasmid-Encoded Virulence-Associated Protein A in Intracellular Survival and Virulence ofRhodococcus equi
Authors: Giguère Steeve , Hondalus Mary K. , Yager Julie A. , Darrah Patricia , Mosser David M. , Prescott John F.
Published: December 2019

Rhodococcus equiis a facultative intracellular pathogen of macrophages and a cause of pneumonia in young horses (foals) and immunocompromised people. Isolates ofR. equifrom pneumonic foals typically contain large, 85- or 90-kb plasmids encoding a highly immunogenic virulence-associated protein (VapA). The objective of this study was to determine the role of the 85-kb plasmid and VapA in the intracellular survival and virulence ofR. equi. Clinical isolates containing the plasmid and expressing VapA efficiently replicated within mouse macrophages in vitro, while plasmid-cured derivatives of these organisms did not multiply intracellularly. An isolate harboring the large plasmid also replicated in the tissues of experimentally infected mice, whereas its plasmid-cured derivative was rapidly cleared. All foals experimentally infected with a plasmid-containing clinical isolate developed severe bronchopneumonia, whereas the foals infected with its plasmid-cured derivative remained asymptomatic and free of visible lung lesions. By day 14 postinfection, lung bacterial burdens had increased considerably in foals challenged with the plasmid-containing clinical isolate. In contrast, bacteria could no longer be cultured from the lungs of foals challenged with the isogenic plasmid-cured derivative. A recombinant, plasmid-cured derivative expressing wild-type levels of VapA failed to replicate in macrophages and remained avirulent for both mice and foals. These results show that the 85-kb plasmid ofR. equiis essential for intracellular replication within macrophages and for development of disease in the native host, the foal. However, expression of VapA alone is not sufficient to restore the virulence phenotype.
The Capsule Supports Survival but Not Traversal ofEscherichia coliK1 across the Blood-Brain Barrier
Authors: Hoffman Jill A. , Wass Carol , Stins Monique F. , Kim Kwang Sik
Published: December 2019

The vast majority of cases of gram-negative meningitis in neonates are caused by K1-encapsulatedEscherichia coli. The role of the K1 capsule in the pathogenesis ofE. colimeningitis was examined with an in vivo model of experimental hematogenousE. coliK1 meningitis and an in vitro model of the blood-brain barrier. Bacteremia was induced in neonatal rats with theE. coliK1 strain C5 (O18:K1) or its K1−derivative, C5ME. Subsequently, blood and cerebrospinal fluid (CSF) were obtained for culture. Viable bacteria were recovered from the CSF of animals infected withE. coliK1 strains only; none of the animals infected with K1−strains had positive CSF cultures. However, despite the fact that their cultures were sterile, the presence of O18E. coliwas demonstrated immunocytochemically in the brains of animals infected with K1−strains and was seen by staining of CSF samples. In vitro, brain microvascular endothelial cells (BMEC) were incubated with K1+and K1−E. colistrains. The recovery of viable intracellular organisms of the K1+strain was significantly higher than that for the K1−strain (P= 0.0005). The recovery of viable intracellular K1−E. colibacteria was increased by cycloheximide treatment of BMEC (P= 0.0059) but was not affected by nitric oxide synthase inhibitors or oxygen radical scavengers. We conclude that the K1 capsule is not necessary for the invasion of bacteria into brain endothelial cells but is responsible for helping to maintain bacterial viability during invasion of the blood-brain barrier.
Early Events in the Pathogenesis of Avian Salmonellosis
Authors: Henderson S. Christine , Bounous Denise I. , Lee Margie D.
Published: December 2019

Salmonellae are gastrointestinal pathogens of man and animals. However, strains that are host-specific avian pathogens are often avirulent in mammals, and those which are nonspecific are commensal in poultry. The objective of this study was to determine whether host specificity was exhibited by bacterial abilities to invade epithelial cells or resist leukocyte killing. In this study, leukocytes isolated from humans and chickens were used to killSalmonellain vitro. BothSalmonella pullorum, an avian-specific serotype, andSalmonella typhimurium, a broad-host-range serotype, were sensitive to killing by polymorphonuclear leukocytes isolated from both species. Both serotypes replicated in cells of the MQ-NCSU avian-macrophage cell line. In contrast,S. pullorumwas noninvasive for cultured epithelial Henle 407, chick kidney, chick ovary, and budgerigar abdominal tumor cells. In the bird challenge, however,S. typhimuriumrapidly caused inflammation of the intestinal mucosa, butS. pullorumpreferentially targeted the bursa of Fabricius prior to eliciting intestinal inflammation.Salmonellaserotypes which cause typhoid fever in mice have been shown to target the gut-associated lymphoid tissue. Observations from this study show thatS. pulloruminitiated a route of infection in chicks comparable to the route it takes in cases of enteric fever.
Resistance of VirulentMycobacterium aviumto Gamma Interferon-Mediated Antimicrobial Activity Suggests Additional Signals for Induction of Mycobacteriostasis
Authors: Flórido Manuela , Gonçalves Ana Sofia , Silva Regina A. , Ehlers Stefan , Cooper Andrea M. , Appelberg Rui
Published: December 2019

The cytokine gamma interferon (IFN-γ) plays a major role in the control ofMycobacterium aviuminfections. We assessed whether the progressive growth of virulent strains ofM. aviumwas associated with alterations in the production of this cytokine as evaluated by reverse transcription-PCR and detection of immunoreactive cytokine in the serum and in spleen homogenates. We found that IFN-γ was induced during infection by a virulent strain ofM. aviumto similar or even higher extents than the levels found during infections by a less virulent strain whose growth was controlled. IFN-γ produced during infection by both mycobacterial strains was partly derived from T cells and led to activation of macrophages, namely, those that were infected. Concomitant with the development of the infection with the virulent strain ofM. aviumthere was an extensive depletion of lymphocytes in the spleen. Thymectomy alone promoted the proliferation of the virulent, but not of the less virulent, strain ofM. avium. Our data indicate that virulent strains ofM. aviumresist the antimicrobial mechanisms of IFN-γ-activated macrophages and raise the possibility that a second, T-cell-dependent signal is required for the effective control of mycobacterial replication inside macrophages.
Correlation of Immunity in Experimental Syphilis with Serum-Mediated Aggregation ofTreponema pallidumRare Outer Membrane Proteins
Authors: Lewinski Michael A. , Miller James N. , Lovett Michael A. , Blanco David R.
Published: December 2019

We have previously shown by freeze-fracture electron microscopy that serum from infection-immune syphilitic rabbits aggregates the low-density membrane-spanningTreponema pallidumrare outer membrane proteins (TROMPs). The purpose of this study was to determine if a relationship could be demonstrated between acquired immunity in experimental rabbit syphilis, serum complement-dependent treponemicidal antibody, and antibody directed against TROMPs as measured by the aggregation of TROMP particles. Three groups ofT. pallidum-infected rabbits were treated curatively with penicillin at 9 days, 30 days, and 6 months postinfection to generate various degrees of immunity to challenge reinfection. Sera from rabbits completely susceptible to localized and disseminated reinfection possessed a low titer of treponemicidal antibody (≤1:1 in killing ≥50% of a treponemal suspension) and showed a correspondingly low level of TROMP aggregation (16.5% of the total number of outer membrane particles counted) similar to normal serum controls (13.4%); the number of particles within these aggregates never exceeded three. Sera from partially immune rabbits, which were susceptible to local reinfection but had no evidence of dissemination, showed an increase in the titer of treponemicidal antibody (1:16) compared to the completely susceptible group (≤1:1). Although no significant increase was observed in the total number of TROMP particles aggregated (18.9%) compared to the number in controls (13.4%), approximately 15% of these aggregates did exhibit a significant increase in the number of particles per aggregate (4 to 5 particles) compared to controls (≤3 particles), indicating a measurable increase in anti-TROMP antibody. Finally, sera from rabbits completely immune to both local and disseminated reinfection possessed both high titers of treponemicidal antibody (1:128) and significant aggregation of TROMP (88.6%); approximately 50% of these aggregates contained four to six particles. The results indicate that complete immunity in experimental rabbit syphilis correlates with antibody that killsT. pallidumand aggregates TROMPs, suggesting that TROMPs are molecules which contribute to the development of acquired immunity.
Enhanced In Vivo Immune Responses to Bacterial Lipopolysaccharide by Exogenous CD40 Stimulation
Authors: Barr Tom A. , Heath Andrew W.
Published: December 2019

The lack of specific T-cell help in immune responses to thymus-independent antigens results in weak, predominantly immunoglobulin M-mediated immunity with little or no memory. In the work presented here we show how the exogenous stimulation of CD40 by monoclonal antibodies can mimic T-cell help, resulting in enhanced immune responses which are protective against bacterial infection.
Development of a System for Expressing Heterologous Genes in the Oral SpirocheteTreponema denticolaand Its Use in Expression of theTreponema pallidum flaAGene
Authors: Chi Bo , Chauhan Sarita , Kuramitsu Howard
Published: December 2019

The present communication describes the construction of a newEscherichia coli-Treponema denticolashuttle vector based on the naturally occurring spirochete plasmid pTS1 and the expression of the heterologousT. pallidum flaAgene from the plasmid inT. denticola. This new shuttle vector system should prove useful in characterizing virulence factors from unculturable pathogenic spirochetes.
Legionella pneumophilaContains a Type II General Secretion Pathway Required for Growth in Amoebae as Well as for Secretion of the Msp Protease
Authors: Hales Laura M. , Shuman Howard A.
Published: December 2019

We report the identification of a set ofLegionella pneumophilagenes that encode products with homology to proteins of the type II general secretion pathway of gram-negative bacteria. A strain containing a deletion-substitution mutation of two of these genes was unable to secrete the Msp protease. This strain was unable to multiply within the free-living amoebaAcanthamoeba castellaniiyet was able to kill HL-60-derived macrophages. Because Msp is not required for growth in amoebae, other proteins which are important for growth in amoebae are likely secreted by this pathway.
Isolation and Characterization of a
sigB
Deletion Mutant of
Staphylococcus aureus
Authors: Nicholas R. O. , Li Tong , McDevitt D. , Marra Andrea , Sucoloski S. , Demarsh P. L. , Gentry D. R.
Published: December 2019

The
sigB
gene of
Staphylococcus aureus
, coding for the alternate sigma factor B, has been deleted by allelic replacement mutagenesis. The mutant grew as well as the parent in vitro, although it was deficient in clumping factor, coagulase, and pigment. In two murine and one rat infection model the mutant showed no reduction in virulence.
Human Monocytic U937 Cells Kill
Salmonella
In Vitro by NO-Independent Mechanisms
Authors: Ekman Päivi , Saarinen Marja , He Qiushui , Virtala Mika , Salmi Marko , Granfors Kaisa
Published: December 2019

Nitric oxide (NO) has a central role in host defense against intracellular microbes. HLA-B27 has been shown to directly modulate host-microbe interaction in vitro, leading to the impaired elimination of
Salmonella
in human monocytic U937 cells. Here, we studied whether impaired elimination of
Salmonella
would result from differences in NO production between HLA-B27- and HLA-A2-transfected U937 cells. Both human monocytic transfectants produced NO equally well and killed
Salmonella
via NO-independent mechanisms.
Anti-CD14 Monoclonal Antibodies Inhibit the Production of Tumor Necrosis Factor Alpha and Interleukin-10 by Human Monocytes Stimulated with Killed and LiveHaemophilus influenzaeorStreptococcus pneumoniaeOrganisms
Authors: van Furth A. Marceline , Verhard-Seijmonsbergen Els M. , Langermans Jan A. M. , van Dissel Jaap T. , van Furth Ralph
Published: December 2019

In previous studies, we have shown that intact, heat-killed, gram-negative bacteria (GNB) and gram-positive bacteria (GPB) can stimulate the production of various proinflammatory and anti-inflammatory cytokines. The objective of the present study was to investigate whether the production of tumor necrosis factor alpha (TNF) and interleukin-10 (IL-10) by human monocytes stimulated by intact heat-killed or liveHaemophilus influenzaeorStreptococcus pneumoniaeis mediated by CD14. Two anti-CD14 monoclonal antibodies (MAbs) were used to study the interaction between human monocytes and bacteria; lipopolysaccharide (LPS) was used to validate the effect of anti-CD14 MAb. MAb 18E12 decreased significantly TNF and IL-10 production upon stimulation with LPS or heat-killed bacteria and TNF production during stimulation by live bacteria. MAb My-4 decreased production of TNF and IL-10 by monocytes stimulated with LPS, IL-10 but not TNF production upon stimulation with heat-killedH. influenzae, and production of neither TNF nor IL-10 upon stimulation withS. pneumoniae. Together, these results led to the conclusion that CD14 is involved in the recognition and stimulation of human monocytes by intact GNB and GPB. Consequentially, the option for adjunctive treatment of severe infections with anti-CD14 MAb is postulated.
Role of Phospholipase D inPasteurella haemolyticaLeukotoxin-Induced Increase in Phospholipase A2Activity in Bovine Neutrophils
Authors: Wang Zuncai , Clarke Cyril R. , Clinkenbeard Kenneth D.
Published: December 2019

The effects ofPasteurella haemolyticaleukotoxin (LKT) on the activity of phospholipase D (PLD) and the regulatory interaction between PLD and phospholipase A2(PLA2) were investigated in assays using isolated bovine neutrophils labeled with tritiated phospholipid substrates of the two enzymes. Exposure of [3H]lysophosphatidylcholine-labeled neutrophils to LKT caused concentration- and time-dependent production of phosphatidic acid (PA), the product of PLD. LKT-induced generation of PA was dependent on extracellular calcium. Both production of PA and metabolism of [3H]-arachidonate ([3H]AA)-labeled phospholipids by PLA2were inhibited when ethanol was used to promote the alternative PLD-mediated transphosphatidylation reaction, resulting in the production of phosphatidylethanol rather than PA. The role of PA in regulation of PLA2activity was then confirmed by means of an add-back experiment, whereby addition of PA in the presence of ethanol restored PLA2-mediated release of radioactivity from neutrophil membranes. Considering the involvement of chemotactic phospholipase products in the pathogenesis of pneumonic pasteurellosis, development and use of anti-inflammatory agents that inhibit LKT-induced activation of PLD and PLA2may improve therapeutic management of the disease.
CTLA-4 Blockade Enhances the Immune Response Induced by Mycobacterial Infection but Does Not Lead to Increased Protection
Authors: Kirman Joanna , McCoy Kathy , Hook Sarah , Prout Melanie , Delahunt Brett , Orme Ian , Frank Anthony , Le Gros Graham
Published: December 2019

The murine immune response to a pulmonary mycobacterial infection is slow to develop, allowing bacterial numbers to increase in the lung for several weeks after infection. We sought to enhance the protective immune response induced duringMycobacterium bovisBCG infection by administering an antibody that blocks the interaction of CTLA-4 with its ligands, CD80 and CD86. We found that injection of anti-CTLA-4 monoclonal antibody (MAb) greatly enhanced and accelerated the immune response, as measured by increased cellularity of the draining mediastinal lymph nodes, and enhanced antigen-inducible proliferation and gamma interferon production by mediastinal lymphocytes in vitro. However, despite the apparently enhanced immune response in the mediastinal lymph node following treatment with anti-CTLA-4 MAb, there was no improvement in clearance of mycobacteria in the lungs, liver, or spleen. Examination of the primary site of infection, the lung, revealed that CTLA-4 blockade had no effect on the number or function of lymphocytes infiltrating the infected lung tissue. Taken together, these data suggest that in vivo CTLA-4 blockade enhances mycobacterial-infection-induced lymphocyte expansion and effector cell cytokine production in the draining lymph node but does not alter the number or function of lymphocytes at the primary site of infection and therefore does not lead to enhanced clearance of the infection.
Variable Carbohydrate Modifications to the Catalytic Chains of the RgpA and RgpB Proteases ofPorphyromonas gingivalisW50
Authors: Curtis Michael A. , Thickett Andrea , Slaney Jennifer M. , Rangarajan Minnie , Aduse-Opoku Joseph , Shepherd Philip , Paramonov Nikolay , Hounsell Elizabeth F.
Published: December 2019

Proteases ofPorphyromonas gingivalisare considered to be important virulence determinants of this periodontal bacterium. Several biochemical isoforms of arginine-specific proteases are derived fromrgpAandrgpB. HRgpA is a heterodimer composed of the catalytic α chain noncovalently associated with a β adhesin chain derived from the C terminus of the initial full-length translation product. The catalytic α chain is also present as a monomer (RgpA) either free in solution or associated with membranes.rgpBlacks the coding region for the adhesin domain present inrgpAand yields only monomeric forms (RgpB) which again may be soluble or membrane associated. In this study, the catalytic chains of this unusual group of enzymes are shown to be differentially modified by the posttranslational addition of carbohydrate. A monoclonal antibody (MAb 1B5) raised to the monomeric RgpA did not react with the corresponding recombinant RgpA α chain expressed inEscherichia colibut was immunoreactive withP. gingivalislipopolysaccharide. MAb 1B5 also reacted with the membrane-associated forms of RgpA and RgpB but not with the heterodimeric HRgpA and the soluble form of RgpB. RgpA treated with denaturants was capable of binding to MAb 1B5 whereas treatment with periodate abolished this binding, suggesting the presence of carbohydrate residues within the epitope. Chemical deglycosylation abolished immunoreactivity with MAb 1B5 and caused a ∼30% reduction in the size of the membrane-associated enzymes. Monosaccharide analysis of HRgpA and RgpA demonstrated 2.1 and 14.4%, respectively, carbohydrate by weight of protein. Furthermore, distinct differences were detected in their monosaccharide compositions, indicating that these protease isoforms are modified not only to different extents but also with different sugars. The variable nature of these additions may have a significant effect on the structure, stability, and immune recognition of these protease glycoproteins.
Molecular Characterization of the Hemin Uptake Locus (
hmu
) from
Yersinia pestis
and Analysis of
hmu
Mutants for Hemin and Hemoprotein Utilization
Authors: Thompson Jan M. , Jones Heather A. , Perry Robert D.
Published: December 2019

Sequence analysis of the hemin uptake locus (
hmu
) of
Yersinia pestis
revealed five genes,
hmuRSTUV
, required for use of hemin and hemoproteins as iron sources. The translated gene products have homologies with proteins of the hemin transport genes of several gram-negative bacteria. Promoters were identified upstream of
hmuP′R
(p1) and upstream of
hmuS
(p2); p1, which contains a Fur box, is regulated by iron and Fur, while p2 exhibits weak, but constitutive, activity. HmuR, which has homology with TonB-dependent outer membrane (OM) receptors, is localized to the OM of
Y. pestis
and is required for utilizing hemin and all hemoproteins under iron-depleted conditions. The proposed ABC transporter, HmuTUV, is necessary for use of hemin, hemin-albumin, and myoglobin, but not hemoglobin, hemoglobin-haptoglobin, or heme-hemopexin, as iron sources. In the absence of HmuTUV, HmuS, a cytoplasmic protein, is involved in use of hemoglobin and heme-hemopexin. In mice, the 50% lethal doses of
Y. pestis
Δ
hmuP′RSTUV
mutants injected subcutaneously or retro-orbitally did not differ from that of the Hmu
+
parent strain. Thus, the
hmu
system is not essential for infection in mice via these routes. Growth studies showed that a Δ
hmuP′RSTUV
mutant could grow in iron-depleted medium containing high concentrations of hemoglobin, suggesting that an Hmu-independent, lower-affinity hemoglobin uptake system may exist.