bluuebunny/crossref_metadata_2025 huggingface dataset:
Nuclear Translocation of NF-κB in Lipopolysaccharide-Treated Macrophages Fails To Correspond to Endotoxicity: Evidence Suggesting a Requirement for a Gamma Interferon-Like Signal
Authors: Denlinger Loren C. , Garis Kristen A. , Sommer Julie A. , Guadarrama Arturo G. , Proctor Richard A. , Bertics Paul J.
Published: December 2019

Elucidation of a signal transduction pathway essential to lipopolysaccharide (LPS)-induced macrophage activation has the capacity to provide new targets for the treatment of septic shock. In this regard, activation of the transcription factor NF-κB is commonly thought to be critical to LPS-stimulated macrophage inflammatory mediator production, although certain immunological, genetic, and molecular evidence suggests that other factors are involved. To address this issue, we hypothesized that the degree of LPS-induced NF-κB mobilization should correlate with the murine endotoxicity of the species of LPS used for in vitro study. Therefore, usingd-galactosamine-sensitized mice, we assessed the lethal potencies of eight LPS preparations fromEscherichia,Salmonella,Klebsiella,Bacteroides,Pseudomonas,Neisseria, andRhodobacterspecies as well as that of the endotoxin substructure lipid X. The lethal potencies of these LPS preparations varied by >160-fold. Treatment of RAW 264.7 cells with the same LPS preparations induced levels of tumor necrosis factor alpha (TNF-α) and NO production that correlated with the LPS 50% lethal dose. The combined analysis of the levels of these two mediators produced in response to LPS in RAW cells was found to be a strong predictor of murine endotoxic lethality. Interestingly, while relatively nontoxic in mice,Rhodobacter capsulatusLPS stimulated RAW cell NF-κB-like DNA binding protein mobilization and TNF-α production to levels comparable to those of more toxic species of LPS but was unable to induce NO generation in RAW cells. These data indicate that neither NF-κB activation nor TNF-α production alone is a dependable predictor of LPS lethality. Additionally, cotreatment of RAW cells with the potent inflammatory mediator ADP had no effect on the ability ofR. capsulatusLPS to stimulate NO production but significantly enhanced induction of NO production by the toxic species of LPS. In contrast, cotreatment of RAW cells or peritoneal macrophages with gamma interferon (IFN-γ) normalized the abilities of both toxic and nontoxic LPS preparations to induce NO production, suggesting that selected preparations of LPS may preferentially generate an IFN-γ-like signal that accounts for enhanced toxicity. In sum, the activation of NF-κB does not correspond to LPS lethality, thereby complicating models of macrophage activation that highlight NF-κB alone as a signal transduction factor necessary for LPS-mediated toxicity.
Enhancement of Susceptibility to Shiga Toxin-Producing
Escherichia coli
O157:H7 by Protein Calorie Malnutrition in Mice
Authors: Kurioka Takaaki , Yunou Yoshihisa , Kita Eiji
Published: December 2019

Infection with Shiga toxin (Stx)-producing enterohemorrhagic
Escherichia coli
is increasing among children. In this study, 5-week-old C57BL/6 mice with protein calorie malnutrition (PCM) that had been fed a 5% protein diet for 2 weeks since ablactation were inoculated intragastrically with 2 × 10
6
CFU of Stx-producing
E. coli
O157:H7. More than 75% of infected mice with PCM died by 10 days postinfection. Infected mice with PCM developed neurologic symptoms 5 days after infection, while well-nourished control mice receiving a 25% protein diet did not. In the intestinal tracts of infected mice with PCM, inoculated
E. coli
O157:H7 multiplied between days 2 and 4 of infection, with a peak of growth at day 4. Although the pathogens were not culturable from the stool after day 7, O157 lipopolysaccharide was detectable in the stool by enzyme-linked immunosorbent assay even after day 8. Stx was detectable in the stool after day 2 of infection and increased in proportion to the growth of inoculated organisms. The maximal production of Stx occurred at 4 days postchallenge, and Stx was detectable in the blood on days 3 to 5. In contrast, well-nourished control mice survived the infection, and all of them remained well even after 3 weeks of infection. In these control mice, inoculated
E. coli
O157:H7 disappeared from the stool before day 3. Stx was not detectable in the stool and blood of infected control mice at any time from day 1 through day 8. Histologically, cerebral hemorrhages seemed to be the cause of acute death of infected mice with PCM. Immunocytochemical staining demonstrated the positive immunoreaction to Stx at the alveus and stratum pyramidale of the hippocampus and in renal tubules of infected malnourished mice. Such immunoreactions were not found in tissues from infected control mice. Histological study of the intestinal epithelium before infection showed that PCM severely affected the development of intestinal epithelia. These findings strongly indicate that PCM-induced nondevelopment of intestinal physical barrier is one of the predisposing factors for infection with Stx-producing
E. coli
O157:H7 in mice and suggest that our mouse model may explain the high incidence of infection with Stx-producing
E. coli
O157:H7 in the children whose intestinal epithelia have not yet completely developed.
Aging and the Immune Response to theHaemophilus influenzaeType b Capsular Polysaccharide: Retention of the Dominant Idiotype and Antibody Function in the Elderly
Authors: Lucas Alexander H. , Reason Donald C.
Published: December 2019

Anti-Haemophilus influenzaeb polysaccharide (Hib PS) antibodies elicited in elderly subjects following conjugate vaccination expressed a light-chain variable-region (VL)-associated idiotype and had functional activities similar to those previously observed in children and younger adults. These findings indicate that advanced age is not accompanied by shifts in the major VLcomponent of the Hib PS-specific repertoire or by diminution of the protective function of antibodies.
MrpB Functions as the Terminator for Assembly of
Proteus mirabilis
Mannose-Resistant
Proteus
-Like Fimbriae
Authors: Li Xin , Mobley Harry L. T.
Published: December 2019

Insertional mutagenesis studies of
mrpB
, a putative pilin-encoding open reading frame of the
mrp
gene cluster, which encodes mannose-resistant
Proteus
-like (MR/P) fimbriae of
Proteus mirabilis
, indicate that MrpB functions as the terminator for fimbrial assembly.
Distribution of Protein Kinase C Isoforms after Infection of Macrophages withLeishmania major
Authors: Pingel Sabine , Wang Zhi-En , Locksley Richard M.
Published: December 2019

We characterized the effects ofLeishmaniainfection on activation-induced translocation of protein kinase C (PKC) isoforms in murine bone marrow-derived macrophages. Although PKC-dependent gene expression was attenuated by infection, the distribution and translocation of PKC isoforms were unaffected. However, subsequent dissociation from membranes was substantially delayed for some isoforms, particularly PKCβ.
Trafficking of Porin-Deficient
Salmonella typhimurium
Mutants inside HeLa Cells:
ompR
and
envZ
Mutants Are Defective for the Formation of
Salmonella
-Induced Filaments
Authors: Mills Scott D. , Ruschkowski Sharon R. , Stein Murry A. , Finlay B. Brett
Published: December 2019

Outer membrane porin genes of
Salmonella typhimurium
, including
ompC
,
ompF
, and
tppB
, are regulated by the products of
ompB
, a two-component regulatory locus encoding OmpR and EnvZ.
S. typhimurium ompR
mutants are attenuated in mice, but to date no one has studied the intracellular trafficking of
S. typhimurium
porin-deficient mutants. In this study, isogenic transposon mutants of
S. typhimurium
with insertions in
ompR
,
envZ
,
ompF
,
ompC
,
ompD
,
osmZ
, and
tppB
were compared with wild-type SL1344 for trafficking in the human epithelial cell line HeLa. We found that
ompR
and
envZ
mutants were reduced or completely inhibited for the formation of
Salmonella
-induced filaments (Sifs). This result was confirmed with an
ompB
deletion mutant. Sifs are tubular structures containing lysosomal glycoprotein which are induced specifically by intracellular
Salmonella
. Genetic analysis showed that the
ompR
mutation could be complemented in
trans
by cloned
ompR
to restore its ability to induce Sifs. In contrast, mutations in the known
ompR
-regulated genes
ompF
,
ompC
, and
tppB
(as well as the
ompR
-independent porin gene,
ompD
) had no effect on Sif formation relative to that of wild-type SL1344, thus indicating that OmpR does not exert its role on these genes to induce Sif formation. The
omp
mutants studied were able to invade and replicate in HeLa cells at levels comparable to those in wild-type SL1344. We conclude that OmpR and EnvZ appear to regulate Sif formation triggered by intracellular
S. typhimurium.
Helicobacter pylori
Glutamine Synthetase Lacks Features Associated with Transcriptional and Posttranslational Regulation
Authors: Garner Rachel M. , Fulkerson John , Mobley Harry L. T.
Published: December 2019

Helicobacter pylori
urease, produced in abundance, is indispensable for the survival of
H. pylori
in animal hosts. Urea is hydrolyzed by the enzyme, resulting in the liberation of excess ammonia, some of which neutralizes gastric acid. The remaining ammonia is assimilated into protein by glutamine synthetase (EC
6.3.1.2
), which catalyzes the reaction: NH
3
+ glutamate + ATP→glutamine + ADP + P
i
. We hypothesized that glutamine synthetase plays an unusually critical role in nitrogen assimilation by
H. pylori
. We developed a phenotypic screen to isolate genes that contribute to the synthesis of a catalytically active urease.
Escherichia coli
SE5000 transformed with plasmid pHP808 containing the entire
H. pylori
urease gene cluster was cotransformed with a pBluescript plasmid library of the
H. pylori
ATCC 43504 genome. A weakly urease-positive 9.4-kb clone, pUEF728, was subjected to nucleotide sequencing. Among other genes, the gene for glutamine synthetase was identified. The complete 1,443-bp
glnA
gene predicts a polypeptide of 481 amino acid residues with a molecular weight of 54,317; this was supported by maxicell analysis of cloned
glnA
expressed in
E. coli
. The top 10 homologs were all bacterial glutamine synthetases, including
Salmonella typhimurium glnA
. The ATP-binding motif GDNGSG (residues 272 to 277) of
H. pylori
GlnA exactly matched and aligned with the sequence in 8 of the 10 homologs. The adenylation site found in the top 10 homologs (consensus sequence, NLYDLP) is replaced in
H. pylori
by NLFKLT (residues 405 to 410). Since the Tyr (Y) residue is the target of adenylation and since the
H. pylori
glutamine synthetase lacks that residue in four strains examined, we conclude that no adenylation occurs within this motif. Cloned
H. pylori glnA
complemented a
glnA
mutation in
E. coli
, and GlnA enzyme activity could be measured spectrophotometrically. In an attempt to produce a GlnA-deficient mutant of
H. pylori
, a kanamycin resistance cassette was cloned into the
Tth
111I site of
H. pylori glnA
. By using the standard technique of allelic exchange mutagenesis, no verifiable glutamine synthetase double-crossover mutant of strain UMAB41 could be isolated, suggesting that the mutation is lethal. We conclude that glutamine synthetase is critical for nitrogen assimilation in
H. pylori
and is active under all physiologic conditions.
Immunization of Cattle by Infection withCowdria ruminantiumElicits T Lymphocytes That Recognize Autologous, Infected Endothelial Cells and Monocytes
Authors: Mwangi Duncan M. , Mahan Suman M. , Nyanjui John K. , Taracha Evans L. N. , McKeever Declan J.
Published: December 2019

Peripheral blood mononuclear cells (PBMC) from immune cattle proliferate in the presence of autologousCowdria ruminantium-infected endothelial cells and monocytes. Endothelial cells required treatment with T-cell growth factors to induce class II major histocompatibility complex expression prior to infection and use as stimulators. Proliferative responses to both infected autologous endothelial cells and monocytes were characterized by expansion of a mixture of CD4+, CD8+, and γδ T cells. However, γδ T cells dominated following several restimulations. Reverse transcription-PCR analysis of cytokine expression byC. ruminantium-specific T-cell lines and immune PBMC revealed weak interleukin-2 (IL-2), IL-4, and gamma interferon (IFN-γ) transcripts at 3 to 24 h after stimulation. Strong expression of IFN-γ, tumor necrosis factor alpha (TNF-α), TNF-β, and IL-2 receptor α-chain mRNA was detected in T-cell lines 48 h after antigen stimulation. Supernatants from these T-cell cultures contained IFN-γ protein. Our findings suggest that in immune cattle aC. ruminantium-specific T-cell response is induced and that infected endothelial cells and monocytes may presentC. ruminantiumantigens to specific T lymphocytes in vivo during infection and thereby play a role in induction of protective immune responses to the pathogen.
Nasopharyngeal Colonization with Nontypeable
Haemophilus influenzae
in Chinchillas
Authors: Yang Yan-ping , Loosmore Sheena M. , Underdown Brian J. , Klein Michel H.
Published: December 2019

Colonization of the nasopharynx by a middle ear pathogen is the first step in the development of otitis media in humans. The establishment of an animal model of nasopharyngeal colonization would therefore be of great utility in assessing the potential protective ability of candidate vaccine antigens (especially adhesins) against otitis media. A chinchilla nasopharyngeal colonization model for nontypeable
Haemophilus influenzae
(NTHI) was developed with antibiotic-resistant strains. This model does not require coinfection with a virus. There was no significant difference in the efficiency of NTHI colonization between adult (1- to 2-year-old) and young (2- to 3-month-old) animals. However, the incidence of middle ear infection following nasopharyngeal colonization was significantly higher in young animals (83 to 89%) than in adult chinchillas (10 to 30%). Chinchillas that had recovered either from a previous middle ear infection caused by NTHI or from an infection by intranasal inoculation with NTHI were completely protected against nasopharyngeal colonization with a homologous strain and were found to be the best positive controls in protection studies. Systemic immunization of chinchillas with inactivated whole-cell preparations significantly protected animals not only against homologous NTHI colonization but also partially against heterologous NTHI infection. In all protected animals, significant serum anti-P6 and anti-HMW antibody responses were observed. The outer membrane P6 and high-molecular-weight (HMW) proteins appear to be promising candidate vaccine antigens to prevent nasopharyngeal colonization and middle ear infection caused by NTHI.
Ambiguous Role of Interleukin-12 inYersinia enterocoliticaInfection in Susceptible and Resistant Mouse Strains
Authors: Bohn Erwin , Schmitt Edgar , Bielfeldt Claudia , Noll Annette , Schulte Ralf , Autenrieth Ingo B.
Published: December 2019

Endogenous interleukin-12 (IL-12) mediates protection againstYersinia enterocoliticain C57BL/6 mice by triggering gamma interferon (IFN-γ) production in NK and CD4+T cells. Administration of exogenous IL-12 confers protection against yersiniae inYersinia-susceptible BALB/c mice but exacerbates yersiniosis in resistant C57BL/6 mice. Therefore, we wanted to dissect the different mechanisms exerted by IL-12 duringYersiniainfections by using different models ofYersinia-resistant and -susceptible mice, including resistant C57BL/6 mice, susceptible BALB/c mice, intermediate-susceptible wild-type 129/Sv mice, 129/Sv IFN-γ-receptor-deficient (IFN-γR−/−) mice and C57BL/6 tumor necrosis factor (TNF) receptor p55 chain-deficient (TNFR p55−/−) mice. IFN-γR−/−mice turned out to be highly susceptible to infection byY. enterocoliticacompared with IFN-γR+/+mice. Administration of IL-12 was protective in IFN-γR+/+mice but not in IFN-γR−/−mice, suggesting that IFN-γR-induced mechanisms are essential for IL-12-induced resistance against yersiniae. BALB/c mice could be renderedYersiniaresistant by administration of anti-CD4 antibodies or by administration of IL-12. In contrast, C57BL/6 mice could be rendered more resistant by administration of transforming growth factor β (TGF-β). Furthermore, IL-12-triggered toxic effects in C57BL/6 mice were abrogated by coadministration of TGF-β. While administration of IL-12 alone increased TNF-α levels, administration of TGF-β or TGF-β plus IL-12 decreased both TNF-α and IFN-γ levels inYersinia-infected C57BL/6 mice. Moreover, IL-12 did not induce toxicity inYersinia-infected TNFR p55−/−mice, suggesting that TNF-α accounts for IL-12-induced toxicity. Taken together, IL-12 may induce different effector mechanisms in BALB/c and C57BL/6 mice resulting either in protection or exacerbation. These results are important for understanding the critical balance of proinflammatory and regulatory cytokines in bacterial infections which is decisive for beneficial effects of cytokine therapy.
Production of Monocyte Chemoattractant Protein 1 in Tuberculosis Patients
Authors: Lin YuanGuang , Gong JianHua , Zhang Ming , Xue Wanfen , Barnes Peter F.
Published: December 2019

To investigate the role of monocyte chemoattractant protein 1 (MCP-1) in the immune response toMycobacterium tuberculosis, we studied MCP-1 production in tuberculosis patients. CD14+blood monocytes from tuberculosis patients spontaneously expressed higher levels of MCP-1 mRNA and protein than CD14+monocytes from healthy tuberculin reactors. MCP-1 production in lymph nodes from tuberculosis patients was also markedly increased. These findings suggest that MCP-1 can contribute to the antimycobacterial inflammatory response by attracting monocytes and T lymphocytes.
Localization of
Chlamydia trachomatis
Heat Shock Proteins 60 and 70 during Infection of a Human Endometrial Epithelial Cell Line In Vitro
Authors: Raulston Jane E. , Paul Terry R. , Knight Stephen T. , Wyrick Priscilla B.
Published: December 2019

Unlike chlamydial lipopolysaccharide, which is released from the developing inclusion to the surface of infected genital epithelial cells, both
Chlamydia trachomatis
heat shock protein (hsp) 60 and 70 antigens remained confined within the inclusion during the course of the chlamydial developmental cycle. Exposure of the infected cells to penicillin to induce a persistent infection or to a lipophilic microbicide did not potentiate secretion or exocytosis of the chlamydial hsp.
Structure-Function Relationship of Antibacterial Synthetic Peptides Homologous to a Helical Surface Region on Human Lactoferrin against
Escherichia coli
Serotype O111
Authors: Chapple Daniel S. , Mason David J. , Joannou Christopher L. , Odell Edward W. , Gant Vanya , Evans Robert W.
Published: December 2019

Lactoferricin includes an 11-amino-acid amphipathic alpha-helical region which is exhibited on the outer surface of the amino-terminal lobe of lactoferrin. Synthetic peptides homologous to this region exhibited potent antibacterial activity against a selected range of both gram-negative and gram-positive bacteria. An analog synthesized with methionine substituted for proline at position 26, which is predicted to disrupt the helical region, abolished antibacterial activity against
Escherichia coli
and considerably reduced antibacterial activity against
Staphylococcus aureus
and an
Acinetobacter
strain. The mode of action of human lactoferrin peptide (HLP) 2 against
E. coli
serotype O111 (NCTC 8007) was established by using flow cytometry, surface plasmon resonance, and transmission electron microscopy. Flow cytometry was used to monitor membrane potential, membrane integrity, and metabolic processes by using the fluorescent probes bis-1,3-(dibutylbarbituric acid)-trimethine oxonol, propidium iodide, and carbonyl cyanide
m
-chlorophenylhydrazone, respectively. HLP 2 was found to act at the cell membrane, causing complete loss of membrane potential after 10 min and of membrane integrity within 30 min, with irreversible damage to the cell as shown by rapid loss of viability. The number of particles, measured by light scatter on the flow cytometer, dropped significantly, showing that bacterial lysis resulted. The peptide was shown to bind to
E. coli
O111 lipopolysaccharide by using surface plasmon resonance. Transmission electron microscopy revealed bacterial distortion, with the outer membrane becoming detached from the inner cytoplasmic membrane. We conclude that HLP 2 causes membrane disruption of the outer membrane, resulting in lysis, and that structural considerations are important for antibacterial activity.
Lipopolysaccharide-Related Stimuli Induce Expression of the Secretory Leukocyte Protease Inhibitor, a Macrophage-Derived Lipopolysaccharide Inhibitor
Authors: Jin Fenyu , Nathan Carl F. , Radzioch Danuta , Ding Aihao
Published: December 2019

Mouse secretory leukocyte protease inhibitor (SLPI) was recently characterized as a lipopolysaccharide (LPS)-induced product of macrophages that antagonizes their LPS-induced activation of NF-κB and production of NO and tumor necrosis factor (TNF) (F. Y. Jin, C. Nathan, D. Radzioch, and A. Ding, Cell 88:417–426, 1997). To better understand the role of SLPI in innate immune and inflammatory responses, we examined the kinetics of SLPI expression in response to LPS, LPS-induced cytokines, and LPS-mimetic compounds. SLPI mRNA was detectable in macrophages by Northern blot analysis within 30 min of exposure to LPS but levels peaked only at 24 to 36 h and remained elevated at 72 h. Despite the slowly mounting and prolonged response, early expression of SLPI mRNA was cycloheximide resistant. Two LPS-induced proteins—interleukin-10 (IL-10) and IL-6—also induced SLPI, while TNF and IL-1β did not. The slow attainment of maximal induction of SLPI by LPS in vitro was mimicked by infection withPseudomonas aeruginosain vivo, where SLPI expression in the lung peaked at 3 days. Two LPS-mimetic molecules—taxol from yew bark and lipoteichoic acid (LTA) from gram-positive bacterial cell walls—also induced SLPI. Transfection of macrophages with SLPI inhibited their LTA-induced NO production. An anti-inflammatory role for macrophage-derived SLPI seems likely based on SLPI’s slowly mounting production in response to constituents of gram-negative and gram-positive bacteria, its induction both as a direct response to LPS and as a response to anti-inflammatory cytokines induced by LPS, and its ability to suppress the production of proinflammatory products by macrophages stimulated with constituents of both gram-positive and gram-negative bacteria.
Escherichia coli
Cytotoxic Necrotizing Factor 1 Effaces Microvilli and Decreases Transmigration of Polymorphonuclear Leukocytes in Intestinal T84 Epithelial Cell Monolayers
Authors: Hofman Paul , Flatau Gilles , Selva Eric , Gauthier Michel , Le Negrate Gaëlle , Fiorentini Carla , Rossi Bernard , Boquet Patrice
Published: December 2019

Cytotoxic necrotizing factor type 1 (CNF1), a 110-kDa toxin-like protein from pathogenic
Escherichia coli
strains, induces an actin cytoskeleton reorganization consisting of the formation of prominent stress fibers by permanent activation of the small GTP-binding protein Rho. Since p21Rho regulates tight-junction permeability and perijunctional actin reorganization in epithelial intestinal cells (A. Nusrat, M. Giry, J. R. Turner, S. P. Colgan, C. A. Parkos, E. Lemichez, P. Boquet, and J. L. Madara, Proc. Natl. Acad. Sci. USA 92:10629–10633, 1995), we used polarized T84 epithelial intestinal cell monolayers to examine whether CNF1 could affect microvillus structure, transepithelial resistance, and polymorphonuclear leukocyte (PMN) transmigration. Incubation of T84 cells with CNF1 did not influence transepithelial resistance, suggesting that barrier function and surface polarity were not affected by the toxin. However, CNF1 effaced intestinal cell microvilli and induced a strong decrease of PMN transepithelial migration in either the luminal-to-basolateral or the basolateral-to-luminal direction. CNF1 could thus be a virulence factor exhibiting a new type of combined activity consisting of effacing of microvilli and occlusion of the epithelial barrier to PMNs. Attenuated transepithelial migration of PMNs could result in the enhanced growth and protection of luminal bacteria.
Protein Kinase A-Mediated Inhibition of Gamma Interferon-Induced Tyrosine Phosphorylation of Janus Kinases and Latent Cytoplasmic Transcription Factors in Human Monocytes byEhrlichia chaffeensis
Authors: Lee Eunjoo H. , Rikihisa Yasuko
Published: December 2019

Ehrlichia chaffeensis, an obligatory intracellular bacterium of monocytes or macrophages, is the etiologic agent of human monocytic ehrlichiosis. Our previous study showed that gamma interferon (IFN-γ) added prior to or at early stage of infection inhibited infection of human monocytes withE. chaffeensis; however, after 24 h of infection, IFN-γ had no antiehrlichial effect. To test whether ehrlichial infection disrupts Janus kinase (Jak) and signal transducer and activator of transcription (Stat) signaling induced by IFN-γ, tyrosine phosphorylation of Stat1, Jak1, and Jak2 inE. chaffeensis-infected THP-1 cells was examined by immunoprecipitation followed by immunoblot analysis. ViableE. chaffeensisorganisms blocked tyrosine phosphorylation of Stat1, Jak1, and Jak2 in response to IFN-γ within 30 min of infection. Similar results were obtained with human peripheral blood monocytes infected withE. chaffeensis. Heat or proteinase K treatment but not periodate treatment ofE. chaffeensisabrogated the inhibitory effect, suggesting that protein factor(s) ofE. chaffeensisis responsible for the inhibition of IFN-γ-induced tyrosine phosphorylation. Preincubation ofE. chaffeensiswith the Fab fragment of dog anti-E. chaffeensisimmunoglobulin G also abrogated the inhibitory effect. On the other hand, monodansylcadaverine, which does not block binding but blocks internalization of ehrlichiae into macrophages, did not have any influence on the tyrosine phosphorylation. These results indicate that ehrlichial binding to host cells is sufficient to inhibit Stat1 tyrosine phosphorylation induced by IFN-γ. Protein kinase A (PKA) activity in THP-1 cells increased approximately 25-fold within 30 min of infection withE. chaffeensis. In THP-1 cells pretreated with a PKA inhibitor, Rp isomer of adenosine 3′,5′-cyclic phosphorothioate,E. chaffeensis-induced inhibition of Stat1 tyrosine phosphorylation was partially abrogated. These results suggest thatE. chaffeensisblocks IFN-γ-induced tyrosine phosphorylation of Jak and Stat through raising PKA activity in THP-1 cells, which may be an important survival mechanism of ehrlichiae within the host cell.
An Epimerase Gene Essential for Capsule Synthesis in
Vibrio vulnificus
Authors: Zuppardo Amy B. , Siebeling Ronald J.
Published: December 2019

The extracellular capsule polysaccharide (CPS) of
Vibrio vulnificus
is a primary virulence factor which allows survival of the bacteria in the human host. To study the genes involved in expression of the capsule, we generated mutants that lost the ability to produce CPS following the insertion of a minitransposon into the genome of an encapsulated, clinical strain of
V. vulnificus
. A genomic region, from one nonencapsulated mutant, containing the transposon and flanking
V. vulnificus
DNA was cloned, and a probe complementary to the chromosomal DNA immediately adjacent to the transposon was used to locate this fragment in the genome of the encapsulated parent strain. The fragment, which contained a putative capsule gene, was cloned and, when supplied in
trans
, complemented the mutation in the nonencapsulated mutant to restore capsule production. In addition, virulence studies, using the 50% lethal dose assay, showed that the restoration of capsule production also restored the virulence of the organism. Sequence analysis of the gene disrupted by the transposon revealed that it matched a nucleotide-sugar epimerase of
Vibrio cholerae
O139, with 75 and 85% identities at the nucleotide and amino acid levels, respectively. In addition, computer analysis recognized epimerases of various organisms as highly similar to the putative epimerase of
V. vulnificus
. Finally, a combination of PCR amplification and Southern blotting showed that this epimerase is common to at least 10 strains of
V. vulnificus
that each express a serologically distinct CPS. Our results indicate that the epimerase gene is essential for capsule expression in
V. vulnificus.
Differential Effects of Myeloperoxidase-Derived Oxidants on
Escherichia coli
DNA Replication
Authors: Rosen Henry , Michel Bryce R. , vanDevanter Donald R. , Hughes James P.
Published: December 2019

The microbicidal myeloperoxidase (MPO)-H
2
O
2
-chloride system strongly inhibits
Escherichia coli
DNA synthesis. Also, cell envelopes from MPO-treated
E. coli
cells lose their ability to interact with hemimethylated DNA sequences of
oriC
, the chromosomal origin of replication, raising the prospect that suppression of DNA synthesis involves impairment of
oriC
-related functions (H. Rosen, et al. Proc. Natl. Acad. Sci. USA, 87:10048–10052, 1990). To evaluate whether origin-specific DNA sequences play a role in the MPO effect on
E. coli
DNA synthesis, plasmid DNA replication was compared to total (chromosomal) DNA replication for six plasmids with three distinct origins of replication. Plasmid pCM700 replication, replicating from
oriC
, was as sensitive to MPO-mediated inhibition as was total (chromosomal) DNA replication. A regression line describing this relationship had a slope of 0.90, and the
r
2
was 0.89. In contrast, the replication activities of three of four non-
oriC
plasmids, pUC19, pACYC184, and pSC101, demonstrated significant early resistance to inhibition by MPO-derived oxidants. The exception to this resistance pattern was plasmid pSP102, which has an origin derived from P1 phage. pSP102 replication declined similarly to that of total DNA synthesis. The regression line for pSP102 replication versus total DNA synthesis had a slope of 0.95, and the
r
2
was 0.92. The biochemical requirements for P1-mediated replication are strikingly similar to those for
oriC
-mediated replication. It is proposed that one of these requirements, common to
oriC
and the P1 origin but not critical to the replication of the other non-
oriC
plasmids, is an important target for MPO-mediated oxidations that mediate the initial decline in
E. coli
chromosomal DNA synthesis.
Genetic and Biochemical Analysis of Mutacin 1140, a Lantibiotic from
Streptococcus mutans
Authors: Hillman J. D. , Novák Jan , Sagura Edy , Gutierrez Juan A. , Brooks T. A. , Crowley Paula J. , Hess M. , Azizi Abdul , Leung K.-P. , Cvitkovitch Dennis , Bleiweis A. S.
Published: December 2019

Streptococcus mutans
JH1000 and its derivatives were previously shown (J. D. Hillman, K. P. Johnson, and B. I. Yaphe, Infect. Immun. 44:141–144, 1984) to produce a low-molecular-weight, broad-spectrum bacteriocin-like inhibitory substance (BLIS). The thermosensitive vector pTV1-OK harboring Tn
917
was used to isolate a BLIS-deficient mutant, DM25, and the mutated gene was recovered by shotgun cloning in
Escherichia coli
. Sequence analysis of insert DNA adjacent to Tn
917
led to the identification of four open reading frames including two (
lanA
and
lanB
) which have substantial homology to the
Staphylococcus epidermidis
structural gene (
epiA
) and a modifying enzyme gene (
epiB
) for biosynthesis of the lantibiotic epidermin, respectively. Although the BLIS activity could not be recovered from broth cultures, high yields were obtained from a solid medium consisting of Todd-Hewitt broth containing 0.5% agarose that was stab inoculated with JH1140 (a spontaneous mutant of JH1000 that produces threefold-elevated amounts of activity). Agar could not substitute for agarose. Chloroform extraction of the spent medium produced a fraction which yielded two major bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The faster-migrating band was absent in chloroform extracts of the mutant, DM25. The amino acid sequence of this band was determined by Edman sequencing and mass spectroscopy. The results showed that it is a lantibiotic, which we have named mutacin 1140, and that the sequence corresponded to that deduced from the
lanA
sequence. We observed a number of similarities of mutacin 1140 to epidermin and an
S. mutans
lantibiotic, B-Ny266, but it appears to have significant differences in the positions of its thioether bridges. It also has other unique features with regard to its leader sequence and posttranslational modification. A proposed structure for mutacin 1140 is presented.
Heterogeneity in Levels of Vacuolating Cytotoxin Gene (
vacA
) Transcription among
Helicobacter pylori
Strains
Authors: Forsyth M. H. , Atherton J. C. , Blaser M. J. , Cover T. L.
Published: December 2019

Broth culture supernatants from Tox
+
Helicobacter pylori
strains induce vacuolation of HeLa cells in vitro and contain VacA in concentrations that are higher than those found in supernatants from Tox
−
H. pylori
strains. To investigate the basis for this phenomenon, we analyzed the transcription of the vacuolating cytotoxin gene (
vacA
) in eight Tox
+
strains (each with a type s1/m1
vacA
genotype) and nine Tox
−
strains (each with a type s2/m2
vacA
genotype). Most of the Tox
+
and Tox
−
strains tested used the same
vacA
transcriptional start point, but Tox
+
strains yielded significantly stronger primer extension signal intensities than did Tox
−
strains (mean densitometry values of 15.8 ± 1.9 versus 8.9 ± 1.7,
P
= 0.0016). Correspondingly, when we introduced
vacA
::
xylE
transcriptional fusions into the chromosomes of a Tox
+
strain (60190) and a Tox
−
strain (86-313), the level of XylE activity in 60190
vacA
::
xylE
was about 30-fold higher than that in 86-313
vacA
::
xylE
. Sequence analysis and promoter exchange experiments indicated that the different levels of
vacA
transcription in these two strains cannot be explained solely by a difference in promoter strength. These data indicate that Tox
+
and Tox
−
H. pylori
strains typically differ not only in the VacA amino acid sequence but also in the level of
vacA
transcription.
Leishmania pifanoiAmastigote Antigen P-4: Epitopes Involved in T-Cell Responsiveness in Human Cutaneous Leishmaniasis
Authors: Haberer Jessica E. , Da-Cruz Alda Maria , Soong Lynn , Oliveira-Neto Manoel P. , Rivas Luis , McMahon-Pratt Diane , Coutinho Sergio G.
Published: December 2019

In experimental murine cutaneous leishmaniasis, the purifiedLeishmania pifanoiamastigote protein P-4 has been shown to induce significant protection against infection. Further, recent studies examining the response of peripheral blood mononuclear cells (PBMC) fromLeishmania braziliensis-infected human patients have demonstrated that the P-4 protein selectively elicits a significant TH1-like response. Because a TH1-like response is associated with cure, epitope studies were conducted to further evaluate the human response to P-4. PBMC from confirmed cutaneous leishmaniasis patients infected withL. braziliensisin Rio de Janeiro, Brazil, an area where the disease is endemic, were examined for T-cell proliferation and/or cytokine production in response to whole-parasite homogenate, isolated P-4 protein, and/or P-4 peptides. Twenty of the 22 patients (91%) examined responded to the native P-4 protein by proliferation and/or gamma interferon (IFN-γ) production. According to the proliferation data, PBMC from 14 patients (64%) were found to respond to the intact P-4 protein (stimulation index of ≥2.5). Fifty-seven percent of the P-4-responsive patients studied responded to at least one of the P-4 peptides; 11 individual peptides were found to elicit a proliferative response. Of 17 patients examined for cytokine production, no PBMC produced detectable interleukin-4 in response to P-4 protein or peptides. However, PBMC from 14 patients (82%) produced significant levels of IFN-γ (≥20 pg/ml) in response to native P-4 protein. Nineteen of the 23 peptides were found to elicit an IFN-γ response from at least two patients. These data indicate that multiple epitopes spanning the entire P-4 molecule are responsible for the TH1-like immune response observed, indicating that the intact P-4 amastigote molecule, rather than selected peptides, may prove to be the most useful for leishmaniasis vaccine development.
Cloning of the Heat Shock Protein 70 (HSP70) Gene ofEhrlichia sennetsuand Differential Expression of HSP70 and HSP60 mRNA after Temperature Upshift
Authors: Zhang Yilan , Ohashi Norio , Rikihisa Yasuko
Published: December 2019

Ehrlichia sennetsuis the causative agent of human Sennetsu ehrlichiosis. Heat shock protein 60 (HSP60) and HSP70 (DnaK) are two major bacterial HSPs, and their interaction modulates the stress response. Previously, we cloned and sequencedgroEand expressedgroELofE. sennetsu. HSP60 (GroEL) was immunogenic and cross-reactive inEhrlichiaspp. The present study was designed to (i) characterize the HSP70 gene of this organism and (ii) determine whether the expression of these two HSPs is inducible upon exposure to heat stress. A gene encoding an HSP70 homolog was isolated and sequenced from a gene library. The ehrlichial HSP70 gene encoded a 637-amino-acid protein, which had an approximate molecular mass of 68,354 Da and which was homologous to DnaK ofEscherichia coli. A DNA sequence resembling −35 and −10 promoter sequences ofE. coli dnaKwas observed upstream of the ehrlichial HSP70 gene. Alignment of the predicted amino acid sequence with that ofE. coliDnaK andBrucella,Salmonella,Borrelia,Chlamydia, andMycobacteriumHSP70s showed 63, 67, 63, 62, 58, and 53% identity, respectively. By reverse transcription-PCR analysis, the mRNA levels of ehrlichial HSP70 and HSP60 were examined after temperature shifts from 28 to 37°C and from 37 to 40°C. HSP70 mRNA induction levels were greater than those of HSP60 mRNA after a 37-to-40°C temperature shift, whereas the reverse was true after a 28-to-37°C temperature shift. Our data suggest that HSP60 and HSP70 may play different roles during transfer from vector temperature to human body temperature and during a febrile condition characteristic of ehrlichial disease. This study also provides a useful model system for examining mRNA expression in obligatory intracellular bacteria.
Factors Affecting the Collagen Binding Capacity of
Staphylococcus aureus
Authors: Gillaspy Allison F. , Lee Chia Y. , Sau Subrata , Cheung Ambrose L. , Smeltzer Mark S.
Published: December 2019

To determine whether the ability of
Staphylococcus aureus
to bind collagen involves an adhesin other than the collagen adhesin encoded by
cna
, we examined the collagen binding capacity (CBC) of 32 strains of
S. aureus
. With only two exceptions, a high CBC corresponded with the presence of
cna
. Both exceptions involved
cna
-positive strains with a low CBC. The first was a single strain (ACH5) that encoded but did not express
cna
. The second were the mucoid strains Smith diffuse and M, both of which encoded and expressed
cna
but bound only minimal amounts of collagen. Analysis of capsule mutants suggests that the reduced CBC observed in the mucoid strains was due to masking of the collagen adhesin on the cell surface and that this masking effect is restricted to heavily encapsulated strains. Differences in the CBC of the remaining
cna
-positive strains were correlated to variations in the level of
cna
transcription and were independent of the number of B domain repeats in the
cna
gene. In all
cna
-positive strains other than ACH5,
cna
transcription was temporally regulated, with
cna
mRNA levels being highest in cells taken from exponentially growing cultures and falling to almost undetectable levels as cultures entered the post-exponential growth phase. The CBC was also highest with cells taken from exponentially growing cultures. Mutation of
agr
resulted in a slight increase in
cna
transcription and a corresponding increase in CBC during the exponential growth phase but did not affect the temporal pattern of
cna
transcription. Mutation of
sar
resulted in a more dramatic increase in CBC and a delay in the post-exponential-phase repression of
cna
transcription. Mutation of both
sar
and
agr
had an additive effect on both CBC and
cna
transcription. We conclude that (i)
cna
encodes the primary collagen-binding adhesin in
S. aureus
, (ii)
sar
is the primary regulatory element controlling expression of
cna
, and (iii) the regulatory effects of
sar
and
agr
on
cna
transcription are independent of the interaction between
sar
and
agr
.
TheSalmonella typhimuriumAhpC Polypeptide Is Not Essential for Virulence in BALB/c Mice but Is Recognized as an Antigen during Infection
Authors: Taylor Patrick D. , Inchley Christopher J. , Gallagher Maurice P.
Published: December 2019

The OxyR regulon is known to mediate protection against oxidizing agents inSalmonella typhimurium. We reported previously thatahp, one of the OxyR-regulated loci, is induced during macrophage interaction (K. P. Francis, P. D. Taylor, C. J. Inchley, and M. P. Gallagher, J. Bacteriol. 179:4046–4048, 1997). We now report on the effects of disruptingahporoxyRon virulence in a BALB/c mouse model. Surprisingly, insertion of a Mudluxderivative withinahpCwas found to result in attenuation, while irreversible inactivation of the locus through insertion of acmlcassette did not. An SL1344 derivative carrying anoxyR::kandisruption was also found to be as virulent as the parental strain. Moreover, both cell-mediated and humoral responses to AhpC were found to develop during the course of infection, probably through T-helper-cell (type I) activation. These results indicate that, although not essential for virulence, AhpC is expressed byS. typhimuriumduring infection of BALB/c mice and constitutes a target for the immune system.
Construction and Vaccine Potential of Acapsular Mutants ofErysipelothrix rhusiopathiae: Use of Excision of Tn916To Inactivate a Target Gene
Authors: Shimoji Yoshihiro , Mori Yasuyuki , Sekizaki Tsutomu , Shibahara Tomoyuki , Yokomizo Yuichi
Published: December 2019

We previously showed that acapsular transposon Tn916mutants ofErysipelothrix rhusiopathiaeare avirulent for mice. In this study, we constructed nonreverting acapsular mutants and examined the vaccine potential of the mutants in mice. A representative acapsular transposon mutant, 33H6, was plated on selective agar containing autoclaved chlortetracycline and quinaldic acid, and two tetracycline-sensitive mutants were obtained. Sequence analysis of chromosomal regions of the mutants in which Tn916had flanked revealed that Tn916had spontaneously excised from the region and that the six new nucleotides, which were presumably inserted with Tn916into 33H6 chromosome, substituted for those present at the insertion site. The mutants were confirmed to be devoid of capsular antigen by Western immunoblotting and were nonvirulent for mice (subcutaneous 50% lethal dose [LD50], >109CFU). The safety and efficacy of acapsular mutants for live vaccines was further studied by using one mutant strain, named YS-1. The YS-1 bacteria were cleared from the skin sites of inoculation, livers, and spleens of the inoculated mice by 7 days after subcutaneous (s.c.) inoculation. Mice immunized s.c. with doses ranging from 2 × 104to 2 × 108CFU of strain YS-1 were completely protected against challenge with 100 LD50of the homologous, highly virulent strain Fujisawa-SmR 21 days postimmunization, and protective immunity conferred by immunization with 2 × 108CFU of the strain lasted for as long as the 3 months of the observation period. In passive immunization experiments, sera collected from mice immunized with strain YS-1 at days 14 and 21 postimmunization provided protection against challenge with Fujisawa-SmR, whereas sera collected at days 4 and 7 did not. Furthermore, specific spleen cell responses toE. rhusiopathiaeantigens were observed in mice immunized with strain YS-1, and cross-protection against the antigenically heterologous bacteriumListeria monocytogeneswas observed at 7 days after immunization in the mice, suggesting that cell-mediated immunity had been induced. These results suggest thatE. rhusiopathiaeYS-1 may be a suitable choice for further studies of vaccine efficacy in swine.
A 20-Kilodalton N-Terminal Fragment of the D15 Protein Contains a Protective Epitope(s) against
Haemophilus influenzae
Type a and Type b
Authors: Yang Yan-ping , Thomas Wayne R. , Chong Pele , Loosmore Sheena M. , Klein Michel H.
Published: December 2019

A conserved 80-kDa minor outer membrane protein, D15, of
Haemophilus influenzae
has been shown to be a protective antigen in laboratory animals against
H. influenzae
type a (Hia) or type b (Hib) infection. To localize the protective B-cell epitope(s) within the D15 protein and to further explore the possibility of using synthetic peptides as vaccine antigens, a 20-kDa N-terminal fragment of D15 protein (truncated D15 [tD15]) was expressed as a fusion protein with glutathione
S
-transferase in
Escherichia coli
. The tD15 moiety was cleaved from glutathione
S
-transferase by using thrombin and purified to homogeneity. The purified soluble tD15 appeared to contain immunodominant protective epitope(s) against Hia and Hib, since rabbit antisera directed against tD15 were capable of protecting infant rats from Hia or Hib bacteremia. The ease of purification of soluble tD15, therefore, makes it a better candidate antigen than the full-length recombinant D15 which is produced as inclusion bodies in
E. coli
. Furthermore, both the purified tD15 fragment and a mixture of tD15-derived peptides spanning amino acid residues 93 to 209 of the mature D15 protein were capable of inhibiting the protection against Hib conferred on infant rats by rabbit anti-tD15 antiserum, indicating that the protective epitopes of D15 may not be conformational. However, the administration of pooled rabbit immune sera raised against the same panel of peptides failed to protect infant rats from Hib infection.
Identification and Sequence Analysis of a 27-Kilobase Chromosomal Fragment Containing a
Salmonella
Pathogenicity Island Located at 92 Minutes on the Chromosome Map of
Salmonella enterica
Serovar Typhimurium LT2
Authors: Wong Kwong-Kwok , McClelland Michael , Stillwell Lisa C. , Sisk Ellen C. , Thurston Sarah J. , Saffer Jeffrey D.
Published: December 2019

Using a genomic approach, we have identified a new
Salmonella
pathogenicity island, SPI-4, which is the fourth
Salmonella
pathogenicity island to be identified. SPI-4 was located at 92 min on the chromosome map and was flanked by the
ssb
and
soxSR
loci. The DNA sequence covering the entire SPI-4 and both boundaries was determined. The size of SPI-4 was about 25 kb and it contains 18 putative open reading frames (ORFs). Three of these ORFs encode proteins that have significant homology with proteins involved in toxin secretion. Another five ORFs encode proteins that have significant homology with hypothetical proteins from
Synechocystis
sp. strain PCC6803 or
Acinetobacter calcoaceticus
. The rest of the ORFs encode novel proteins, one of which has five membrane-spanning domains. SPI-4 is likely to carry a type I secretion system involved in toxin secretion. Furthermore, a previously identified locus (
ims98
), which is required for intramacrophage survival, was also mapped within the SPI-4 region. These findings suggested that SPI-4 is needed for intramacrophage survival.
Characterization of MGC2, a
Mycoplasma gallisepticum
Cytadhesin with Homology to the
Mycoplasma pneumoniae
30-Kilodalton Protein P30 and
Mycoplasma genitalium
P32
Authors: Hnatow Linda L. , Keeler Calvin L. , Tessmer Laura L. , Czymmek Kirk , Dohms John E.
Published: December 2019

A second cytadhesin-like protein, MGC2, was identified in the avian respiratory pathogen
Mycoplasma gallisepticum
. The 912-nucleotide
mgc2
gene encodes a 32.6-kDa protein with 40.9 and 31.4% identity with the
M. pneumoniae
P30 and
M. genitalium
P32 cytadhesins, respectively. Functional studies with reverse transcription-PCR, immunoblotting, double-sided immunogold labeling, and attachment inhibition assays demonstrated homology to the human mycoplasmal P30 and P32 cytadhesins. These findings suggest that there is a family of cytadhesin genes conserved among pathogenic mycoplasmas infecting widely divergent hosts.