bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

ABSTRACT

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

ABSTRACTEhrlichia 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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACTIn 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

ABSTRACTEhrlichia 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

ABSTRACT

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

ABSTRACTThe 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

ABSTRACTWe 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

ABSTRACT

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.