bluuebunny/crossref_metadata_2025 huggingface dataset:
Vaccine Potential of Attenuated Mutants ofCorynebacterium pseudotuberculosisin Sheep
Authors: Simmons Cameron P. , Dunstan Sarah J. , Tachedjian Mary , Krywult Jolanta , Hodgson Adrian L. M. , Strugnell Richard A.
Published: December 2019

Corynebacterium pseudotuberculosis, a gram-positive facultative intracellular bacterial pathogen, is the etiological agent of the economically important disease caseous lymphadenitis (CLA) in both sheep and goats. Attenuated mutants ofC. pseudotuberculosishave the potential to act as novel vaccines against CLA and as veterinary vaccine vectors. In this report, we have assessed the virulence of botharoQandpldmutants ofC. pseudotuberculosisin sheep and concurrently their capacity to act as vaccines against homologous challenge. The results suggest thataroQmutants ofC. pseudotuberculosisare attenuated with regard to both lymph node persistence and vaccination site reactogenicity. Immunologically,aroQmutants failed to elicit detectable specific gamma interferon (IFN-γ)-secreting lymphocytes and induced low levels of antibodies toC. pseudotuberculosisculture supernatant antigens. Following subcutaneous vaccination, the immune responses induced byaroQmutants did not protect sheep from infection with the wild-type strain but did appear to reduce the clinical severity of disease resulting from challenge. Conversely, an attenuatedC. pseudotuberculosisstrain expressing an enzymatically inactive phospholipase D exotoxin, when used as a vaccine, elicited a protective immune response. Protection appeared to correlate with in vivo persistence of the vaccine strain, the induction of IFN-γ-secreting lymphocytes, and relatively high levels of antibodies to culture supernatant antigens. The results suggest thataroQmutants ofC. pseudotuberculosismay be overly attenuated for use as a CLA vaccines or as vaccine vectors.
Lactoferrin Inhibits the Endotoxin Interaction with CD14 by Competition with the Lipopolysaccharide-Binding Protein
Authors: Elass-Rochard Elisabeth , Legrand Dominique , Salmon Valerie , Roseanu Anca , Trif Mihaela , Tobias Peter S. , Mazurier Joel , Spik Genevieve
Published: December 2019

Human lactoferrin (hLf), a glycoprotein released from neutrophil granules during inflammation, and the lipopolysaccharide (LPS)-binding protein (LBP), an acute-phase serum protein, are known to bind to the lipid A of LPS. The LPS-binding sites are located in the N-terminal regions of both proteins, at amino acid residues 28 to 34 of hLf and 91 to 108 of LBP. Both of these proteins modulate endotoxin activities, but they possess biologically antagonistic properties. In this study, we have investigated the competition between hLf and recombinant human LBP (rhLBP) for the binding ofEscherichia coli055:B5 LPS to the differentiated monocytic THP-1 cell line. Our studies revealed that hLf prevented the rhLBP-mediated binding of LPS to the CD14 receptor on cells. Maximal inhibition of LPS-cell interactions by hLf was raised when both hLf and rhLBP were simultaneously added to LPS or when hLf and LPS were mixed with cells 30 min prior to the incubation with rhLBP. However, when hLf was added 30 min after the interaction of rhLBP with LPS, the binding of the rhLPS-LBP complex to CD14 could not be reversed. These observations indicate that hLf competes with rhLBP for the LPS binding and therefore interferes with the interaction of LPS with CD14. Furthermore, experiments involving competitive binding of the rhLBP-LPS complex to cells with two recombinant mutated hLfs show that in addition to residues 28 to 34, another basic cluster which contains residues 1 to 5 of hLf competes for the binding to LPS. Basic sequences homologous to residues 28 to 34 of hLf were evidenced on LPS-binding proteins such as LBP, bactericidal/permeability-increasing protein, andLimulusanti-LPS factor.
Dynamics of the Murine Humoral Immune Response toNeisseria meningitidisGroup B Capsular Polysaccharide
Authors: Colino Jesús , Outschoorn Ingrid
Published: December 2019

Immunization withNeisseria meningitidisgroup B capsular polysaccharide (CpsB) elicited responses in adult mice that showed the typical dynamic characteristics of the response to a thymus-independent antigen, in contrast to the thymus-dependent behavior of antibody responses to CpsC. The former had a short latent period and showed a rapid increase in serum antibodies that peaked at day 5, and immunoglobulin M (IgM) was the major isotype even though IgG (mainly IgG2a and IgG2b) was also detectable. This response was of short duration, and the specific antibodies were rapidly cleared from the circulation. The secondary responses were similar in magnitude, kinetics, IgM predominance, and IgG distribution. Nevertheless, a threefold IgG increase, a correlation between IgM and IgG levels, and dose-dependent secondary responses were observed. Hyperimmunization considerably reinforced these responses: 10-fold for IgM and 300-fold for IgG. This favored isotype switch was accompanied by a progressive change in the subclass distribution to IgG3 (62%) and IgG1 (28%), along with the possible generation of B-cell memory. The results indicate that CpsB is being strictly thymus independent and suggest that unresponsiveness to purified CpsB is due to tolerance.
Analysis of theYersinia pestisV Protein for the Presence of Linear Antibody Epitopes
Authors: Pullen Jeffrey K. , Anderson George W. , Welkos Susan L. , Friedlander Arthur M.
Published: December 2019

The V protein expressed by pathogenicYersinia pestisis an important virulence factor and protective immunogen. The presence of linear B-cell epitopes in the V protein was investigated by using a series of 17 overlapping linear peptides. Groups of 10 mice were immunized intraperitoneally with 30 μg of each peptide on days 0, 30, and 60. Although the V protein-specific antibody response to the peptides varied, most of the peptides elicited high antibody titers. The immunized mice were challenged subcutaneously with 60 50% lethal doses (LD50) (1 LD50= 1.9 CFU) of a virulentY. pestisstrain, CO92. None of the peptide-immunized mice survived challenge. The animals immunized with the V protein were completely protected against challenge. The immunogenicity of some of the V peptides was increased by conjugating them to keyhole limpet hemocyanin. Only one peptide (encompassing amino acids 1 to 30) conjugate demonstrated some protection; the others were not protective. In additional experiments, V peptides that reacted well with sera from mice survivingY. pestisinfection were combined and used to immunize mice. Although the combined peptides appeared to be very immunogenic, they were not protective. Therefore, the protective B-lymphocyte epitope(s) in the V protein is most likely to be conformational.
Identification and Characterization of the PutP Proline Permease That Contributes to In Vivo Survival ofStaphylococcus aureusin Animal Models
Authors: Schwan William R. , Coulter Silvija N. , Ng Eva Y. W. , Langhorne Michael H. , Ritchie Heather D. , Brody Linnea L. , Westbrock-Wadman Shannon , Bayer Arnold S. , Folger Kim R. , Stover C. Kendall
Published: December 2019

Staphylococcus aureusis an important pathogen of humans and other animals, causing bacteremia, abscesses, endocarditis, and other infectious syndromes. A signature-tagged mutagenesis (STM) system was adapted for use in studying the genes required for in vivo survival ofS. aureus. An STM library was ultimately created inS. aureusRN6390, with Tn917being used to create the transposon mutations. Pools ofS. aureusRN6390 mutants were screened in mouse abscess, bacteremia, and wound infection models for growth attenuation after in vivo passage. One of the mutants that was identified displayed marked attenuation following large-pool screening in all three animal models, which was confirmed in bacteremia and endocarditis models of infection with a smaller pool of mutants. Sequence analysis of the entire open reading frame showed a 99% identity to the high-affinity proline permease (putP) gene characterized in another strain ofS. aureus. In wound and murine abscess infection models, theputPmutant was approximately 10-fold more attenuated than was wild-type strain RN6390. AnotherS. aureusstrain transduced with theputPmutation also displayed an attenuated phenotype after passage in the wound model. A [3H]proline uptake assay showed that less proline was specifically transported into theputPmutant than into strain RN6390. The reduced viability of the bacteria possessing the mutation in theS. aureushigh-affinity proline permease suggests that proline scavenging by the bacteria is important for in vivo growth and proliferation and that analogs of proline may serve as potential antistaphylococcal therapeutic agents.
Activation of Bovine Lymphocyte Subpopulations by Staphylococcal Enterotoxin C
Authors: Ferens Witold A. , Davis William C. , Hamilton Mary Jo , Park Yong H. , Deobald Claudia F. , Fox Lawrence , Bohach Gregory
Published: December 2019

Staphylococcus aureusis a major mastitis-causing pathogen in cattle. The chronic nature of bovine staphylococcal mastitis suggests that some products or components ofS. aureusmay interfere with the development of protective immunity. One class of molecules that could be involved are superantigens (SAgs). Although a significant number of mastitis isolates produce SAgs, the effect of these molecules on the bovine immune system is unresolved. To determine if immunosuppression caused by SAgs could play a role in pathogenesis, we monitored bovine lymphocytes exposed to staphylococcal enterotoxin C1 (SEC1). Activation of bovine lymphocytes by either SEC1 or concanavalin A (ConA) was influenced by the γδ/αβ T-cell ratio in the culture. Compared to ConA-induced stimulation, cultures stimulated with SEC1 generated small numbers of CD4+αβ T cells expressing high levels of interleukin-2 receptor α chain (IL-2Rα) and major histocompatibility complex class II (MHCII), suggesting that SAg exposure does not lead to full activation of these cells. This state of partial activation was most pronounced in cultures with a high γδ/αβ ratio. In contrast, significant numbers of CD8+αβ T cells expressed high levels of IL-2Rα and MHCII, regardless of the γδ/αβ ratio and the stimulant used. CD8+blasts in cultures stimulated with SEC1 also expressed another activation marker, ACT3, previously detected predominantly on thymocytes and CD4+T cells. Although γδ CD2−and CD2+T cells expressed MHCII and IL-2Rα following stimulation with SEC1, only a few cells increased to blast size, suggesting that they were only partially activated. The results suggest ways in which SAgs might facilitate immunosuppression that promotes the persistence of bacteria in cattle and contributes to chronic intramammary infection.
Coiling Phagocytosis Discriminates between Different Spirochetes and Is Enhanced by Phorbol Myristate Acetate and Granulocyte- Macrophage Colony-Stimulating Factor
Authors: Rittig M. G. , Jagoda J. C. , Wilske B. , Murgia R. , Cinco M. , Repp R. , Burmester G. R. , Krause A.
Published: December 2019

The mechanisms involved in coiling phagocytosis are not yet known, and it is not even clear whether this phenomenon is either an incidental event or a specific response. Therefore, the phagocytic uptake ofBorrelia burgdorferiand other spirochetes by human monocytes in vitro was used to investigate the involvement of both sides—microbes and phagocytes—in coiling phagocytosis. As seen with electron microscopy, morphologically similarBorrelia,LeptospiraandTreponemastrains induced markedly different frequencies of coiling phagocytosis. The monocytes used coiling phagocytosis for both live (motile) and killed (nonmotile)B. burgdorferi, but pseudopod coils were observed neither with fragmentedB. burgdorferinor with cell-free supernatant fromB. burgdorfericultures. Investigation of the relationship of coiling phagocytosis with other pseudopod-based cellular mechanisms revealed that the use of bioreagents that inhibit conventional phagocytosis also inhibited coiling phagocytis but did not affect membrane ruffling. Bioreagents that increase membrane ruffling did not affect phagocytosis ofB. burgdorferi, except for granulocyte-macrophage colony-stimulating factor and phorbol myristate acetate, which increased coiling phagocytosis selectively. These results demonstrate that coiling phagocytosis is not induced by microbial motility, viability, or a certain morphology and that it is not a random event. Rather, it is a selective uptake mechanism actively driven by the phagocytes. However, whether coiling phagocytosis represents an independent alternative to conventional phagocytosis or, alternatively, a fault in conventional phagocytosis remains to be determined.
Apoptosis of Renal Cortical Cells in the Hemolytic-Uremic Syndrome: In Vivo and In Vitro Studies
Authors: Karpman Diana , Håkansson Anders , Perez Maria-Thereza R. , Isaksson Christina , Carlemalm Eric , Caprioli Alfredo , Svanborg Catharina
Published: December 2019

This study examined apoptotic cell death associated with Shiga-like toxin (Stx)-producing
Escherichia coli
. Renal cortices from three children with postenteropathic hemolytic-uremic syndrome (HUS) and from mice infected with
E. coli
O157:H7 and pediatric renal tubular epithelial cells stimulated with Stx and
E. coli
O157:H7 extracts were examined for apoptotic changes. Apoptotic cells were detected by terminal dUTP nick end labeling of tubuli and glomeruli from HUS patients and from mice inoculated with Stx-2-positive and Stx-negative strains. Apoptosis was more extensive and severe ultramorphological nuclear and cytoplasmic changes were seen in the Stx-2-positive group. Stx caused DNA fragmentation and ultramorphological changes indicating apoptosis in cultured pediatric tubular cells. DNA fragmentation increased when cells were prestimulated with tumor necrosis factor alpha. Polymyxin extracts from Stx-2-positive and Stx-negative strains induced DNA fragmentation, but only extracts from Stx-2-positive strains caused ultramorphological changes and extensive DNA fragmentation. The results indicate that HUS is accompanied by increased apoptosis of kidney cells and that bacterial factors, possibly together with host cytokines in vivo, may activate apoptotic tissue injury.
Filamentous Actin Disruption and Diminished Inositol Phosphate Response in Gingival Fibroblasts Caused by
Treponema denticola
Authors: Yang Po Fong , Song Meja , Grove David A. , Ellen Richard P.
Published: December 2019

Previous reports have shown that
Treponema denticola
causes rearrangement of filamentous actin (F-actin) in human gingival fibroblasts (HGF). The purpose of this investigation was to determine the effect of
T. denticola
on the generation of inositol phosphates (IPs) in relation to a time course for F-actin disruption in HGF. Cultured HGF were exposed to washed cells of
T. denticola
ATCC 35405 for 140 min. Changes in the fluorescence intensity of rhodamine-phalloidin-labeled F-actin in serial optical sections of single HGF were quantified by confocal microscopy image analysis. The percentage of cells with stress fiber disruption was also determined by fluorescence microscopy. Challenge with
T. denticola
caused a significant reduction in F-actin within the first hour, especially at the expense of F-actin in the ventral third of the cells, and a significant increase in the percentage of HGF with altered stress fiber patterns. Significant concentration-dependent disruption of stress fibers was also caused by HGF exposure to a Triton X-100 extract of
T. denticola
outer membrane (OM). IPs were measured by a radiotracer assay based on the incorporation of
myo
-[
3
H]inositol into IPs in HGF incubated with LiCl to inhibit endogenous phosphatases. HGF challenge with several strains of
T. denticola
and the OM extract of
T. denticola
ATCC 35405 resulted in a diminished accumulation of radiolabeled IPs relative to both 15 and 1% fetal bovine serum, which served as strongly positive and background control agonists, respectively. The significantly diminished IP response to
T. denticola
ATCC 35405 occurred within 60 min, concomitant with significant reduction of total F-actin and disruption of stress fibers. Pretreatment with the proteinase inhibitor phenylmethylsulfonyl fluoride, which had previously been found to block
T. denticola
’s degradation of endogenous fibronectin and detachment of HGF from the extracellular matrix, had little effect on F-actin stress fiber disruption and the IP response. Therefore, in addition to its major surface chymotrypsin-like properties,
T. denticola
expresses cytopathogenic activities that diminish the generation of IPs during the time course associated with significant cytoskeletal disruption in fibroblasts.
Treponema denticola
Outer Membrane Inhibits Calcium Flux in Gingival Fibroblasts
Authors: Ko Kevin S.-C. , Glogauer Michael , McCulloch Christopher A. G. , Ellen Richard P.
Published: December 2019

Treponema denticola
is a cultivable oral spirochete which perturbs the cytoskeleton in cultured cells of oral origin, but intracellular signalling pathways by which it affects actin assembly are largely unknown. As the outer membrane (OM) of
Treponema denticola
disrupts actin-dependent processes that normally require precise control of intracellular calcium, we studied the effects of an OM extract on internal calcium release, ligand-gated and calcium release-activated calcium channels, and related mechanosensitive cation fluxes in human gingival fibroblasts (HGF). Single-cell ratio fluorimetry demonstrated that in resting cells loaded with Fura-2, baseline intracellular Ca
2+
concentration ([Ca
2+
]
i
) was not affected by treatment with OM extract, but normal spontaneous [Ca
2+
]
i
oscillations were dramatically increased in frequency for 20 to 30 min followed by complete blockade. OM extract inhibited ATP-induced and thapsigargin-induced release of calcium from intracellular stores by 40 and 30%, respectively. Addition of Ca
2+
to the extracellular pool following depletion of intracellular Ca
2+
by thapsigargin and extracellular Ca
2+
by EGTA yielded 59% less replenishment of [Ca
2+
]
i
in OM extract-treated than in control HGF. In cells loaded with collagen-coated ferric oxide beads to stimulate integrin-dependent calcium release, baseline [Ca
2+
]
i
was nearly doubled but was not significantly different in control and OM extract-treated cells. Magnetically generated tensile forces on the beads induced >300% increases of [Ca
2+
]
i
above baseline. Cells preincubated with OM extract exhibited dose-dependent and time-dependent reductions in stretch-induced [Ca
2+
]
i
transients, which were due to neither loss of beads from the cells nor cell death. The
T. denticola
OM inhibitory activity was eliminated by heating the OM extract to 60°C and by boiling but not by phenylmethylsulfonyl fluoride treatment. Thus nonlipopolysaccharide, nonchymotrypsin, heat-sensitive protein(s) in
T. denticola
OM can evidently inhibit both release of calcium from internal stores and uptake of calcium through the plasma membrane, possibly by interference with calcium release-activated channels.
B-Cell Epitopes and Quantification of the ESAT-6 Protein ofMycobacterium tuberculosis
Authors: Harboe Morten , Malin Adam S. , Dockrell Hazel S. , Gotten Wiker Harald , Ulvund Gunni , Holm Arne , Jørgensen Mikala Clok , Andersen Peter
Published: December 2019

ESAT-6 is an important T-cell antigen recognized by protective T cells in animal models of infection withMycobacterium tuberculosis. In an enzyme-linked immunosorbent assay (ELISA) with overlapping peptides spanning the sequence of ESAT-6, monoclonal antibody HYB76-8 reacted with two peptides in the N-terminal region of the molecule. Assays with synthetic truncated peptides allowed a precise mapping of the epitope to the residues EQQWNFAGIEAAA at positions 3 to 15. Hydrophilicity plots revealed one hydrophilic area at the N terminus and two additional areas further along the polypeptide chain. Antipeptide antibodies were generated by immunization with synthetic 8-mer peptides corresponding to these two regions coupled to keyhole limpet hemocyanin. Prolonged immunization with a 23-mer peptide (positions 40 to 62) resulted in the formation of antibodies reacting with the peptide as well as native ESAT-6. A double-antibody ELISA was then developed with monoclonal antibody HYB76-8 as a capture antibody, antigen for testing in the second layer, and antipeptide antibody in the third layer. The assay was suitable for quantification of ESAT-6 inM. tuberculosisantigen preparations, showing no reactivity withM. bovisBCG Tokyo culture fluid, used as a negative control, or with MPT64 or antigen 85B, previously shown to cross-react with HYB76-8. This capture ELISA permitted the identification of ESAT-6 expression from vaccinia virus constructs containing theesat-6gene; this expression could not be identified by standard immunoblotting.
Inoculum Composition and
Salmonella
Pathogenicity Island 1 Regulate M-Cell Invasion and Epithelial Destruction by
Salmonella typhimurium
Authors: Clark M. Ann , Hirst Barry H. , Jepson Mark A.
Published: December 2019

In the mouse model of
Salmonella typhimurium
infection, the specialized antigen-sampling intestinal M cells are the primary route of
Salmonella
invasion during the early stages of infection. Under certain experimental conditions, M-cell invasion is accompanied by M-cell destruction and loss of adjacent regions of the follicle-associated epithelium (FAE), although the conditions responsible for expression of the cytotoxic phenotype in a proportion of previous studies have not been defined. In the present study, we have demonstrated that the cytotoxic effect exerted by wild-type
S. typhimurium
on mouse Peyer’s patch FAE is dependent on the inoculum composition. We have also demonstrated that the extent of FAE destruction correlates with the extent of M-cell invasion. Bacteria inoculated in Luria-Bertani (LB) broth induce extensive FAE loss and exhibit efficient M-cell invasion, whereas bacteria inoculated in phosphate-buffered saline fail to induce significant FAE disruption and invade M cells at significantly lower levels. Similarly, inoculation in LB significantly enhances invasion of Madin-Darby canine kidney cells by wild-type
S. typhimurium
. Mutants defective for expression of
invA
, a component of
Salmonella
pathogenicity island 1 which is vital for efficient invasion of cultured cells, fail to induce FAE destruction and, when inoculated in LB, are attenuated for M-cell invasion. Variation in
inv
gene expression is, therefore, one possible mechanism by which inoculate composition may regulate the virulence of wild-type
S. typhimurium
. Our findings suggest that the composition of the gut luminal contents may be critical in determining the outcome of naturally acquired
Salmonella
infections and that both vaccine formulation and dietary status of vaccine recipients may significantly affect the efficacy and safety of live
Salmonella
oral vaccine delivery systems.
Protective Role of Gamma Interferon in Experimental Pulmonary Paracoccidioidomycosis
Authors: Cano Luz E. , Kashino Suely S. , Arruda Celina , André Denise , Xidieh Cynthia F. , Singer-Vermes Lucia M. , Vaz Celidéia A. C. , Burger Eva , Calich Vera L. G.
Published: December 2019

We have developed a murine model of pulmonary infection byParacoccidioides brasiliensisin which resistance was associated with immunological activities governed by gamma interferon (IFN-γ). To better characterize this model, we measured type 1 and type 2 cytokines in the lungs and investigated the effect of endogenous IFN-γ depletion by monoclonal antibodies in the course of infection of susceptible (B10.A) and resistant (A/Sn) mice. At weeks 4 and 8 after infection, lungs from susceptible animals presented levels of IFN-γ, interleukin-4 (IL-4), IL-5, and IL-10 higher than those in resistant mice. In both mouse strains, neutralization of endogenous IFN-γ induced exacerbation of the pulmonary infection, earlier fungal dissemination to the liver and spleen, impairment of the specific cellular immune response resulting in significantly lower delayed-type hypersensitivity reactions, and increased levels of immunoglobulin G1 (IgG1)- and IgG2b-specific antibodies. Histopathological analysis demonstrated that depletion of IFN-γ changes the focal granulomatous lesions found in the lungs of B10.A and A/Sn mice into coalescent granulomata which destroy the pulmonary architecture. These results suggest that irrespective of the mouse strain, IFN-γ plays a protective role and that this cytokine is one major mediator of resistance againstP. brasiliensisinfection in mice.
Adherence of
Streptococcus pneumoniae
to Human Bronchial Epithelial Cells (BEAS-2B)
Authors: Adamou John E. , Wizemann Theresa M. , Barren Philip , Langermann Solomon
Published: December 2019

Pneumococcal adherence to alveolar epithelial cells and nasopharyngeal epithelial cells has been well characterized. However, the interaction of
Streptococcus pneumoniae
with bronchial epithelial cells has not been studied. We have now shown that pneumococci bind specifically to a human bronchial epithelial cell line (BEAS-2B cells). Pneumococci adhered to BEAS-2B cells in a time- and dose-dependent manner. These results suggest that the bronchial epithelium may serve as an additional site of attachment for pneumococci and demonstrate the utility of the BEAS-2B cell line for studying mechanisms of pneumococcal infection.
Use of Representational Difference Analysis To Identify Genomic Differences between Pathogenic Strains of
Vibrio cholerae
Authors: Calia Kerstin E. , Waldor Matthew K. , Calderwood Stephen B.
Published: December 2019

Representational difference analysis (RDA) is a recently developed technique used for amplifying genetic differences between two closely related genomes. We compared RDA and a modified version of RDA to examine genomic differences between the two
Vibrio cholerae
serogroups that cause epidemic cholera, O1 and O139, and between the two biotypes of the O1 serogroup. With both techniques, we recovered several sequences known to be found only in
V. cholerae
O139 but absent in its presumed progenitor,
V. cholerae
O1 El Tor. A greater number of unique fragments were generated in comparing the two
V. cholerae
O1 biotypes, consistent with the probable greater genetic differences between the two biotypes.
The
Pseudomonas aeruginosa
Flagellar Cap Protein, FliD, Is Responsible for Mucin Adhesion
Authors: Arora Shiwani K. , Ritchings Bruce W. , Almira Ernesto C. , Lory Stephen , Ramphal Reuben
Published: December 2019

Mucin-specific adhesion of
Pseudomonas aeruginosa
plays an important role in the initial colonization of this organism in the airways of cystic fibrosis patients. We report here that the flagellar cap protein, FliD, participates in this adhesion process. A polar chromosomal insertional mutation in the
P. aeruginosa fliD
gene made this organism nonadhesive to mucin in an in vitro mucin adhesion assay. The adhesive phenotype was restored by providing the
fliD
gene alone on a multicopy plasmid, suggesting involvement of this gene in mucin adhesion of
P. aeruginosa
. Further supporting this observation, the in vitro competition experiments demonstrated that purified FliD protein inhibited the mucin adhesion of nonpiliated
P. aeruginosa
PAK-NP, while the same concentrations of PilA and FlaG proteins of
P. aeruginosa
were ineffective in this function. The regulation of the
fliD
gene was studied and was found to be unique in that the transcription of the
fliD
gene was independent of the flagellar sigma factor ς
28
. Consistent with this finding, no ς
28
binding sequence could be identified in the
fliD
promoter region. The results of the β-galactosidase assays suggest that the
fliD
gene in
P. aeruginosa
is regulated by the newly described transcriptional regulator FleQ and the alternate sigma factor ς
54
(RpoN).
Nonopsonic Phagocytosis of Group C
Neisseria meningitidis
by Human Neutrophils
Authors: Estabrook Michele M. , Zhou Daoguo , Apicella Michael A.
Published: December 2019

Although complement-mediated bactericidal activity in serum has long been known to be very important in host defense against
Neisseria meningitidis
, recent studies have shown that opsonic phagocytosis by neutrophils is also important. The purpose of this study was to determine if endemic group C
N. meningitidis
strains were susceptible to nonopsonic (complement- and antibody-independent) phagocytosis by human neutrophils, which is a well-described phenomenon for
Neisseria gonorrhoeae
. Gonococci that possess one or more of a group of heat-modifiable outer membrane proteins (called opacity-associated [Opa] proteins) are phagocytosed by neutrophils in the absence of serum. We found that four serogroup C meningococcal strains bearing the lacto-
N
-neotetraose (LNnT) structure on lipooligosaccharide (LOS) were phagocytosed by neutrophils in the absence of antibody and active complement. Confocal microscopy confirmed that the organisms were internalized by neutrophils. This susceptibility was not restricted to carrier isolates, since two of the strains were cultured from blood or cerebrospinal fluid. All four strains expressed Opa protein and had relatively less endogenous LOS and capsule sialylation compared to six strains that were resistant to this type of phagocytosis. Nonopsonic phagocytosis of two of the four strains was inhibited by exogenous sialylation of LOS LNnT and the binding of monoclonal antibody to LNnT. However, an isogenic mutant that lacked the LNnT structure was fully susceptible to nonopsonic phagocytosis. We conclude that group C meningococci can be phagocytosed by neutrophils in the absence of antibody and active complement possibly by two different mechanisms. Expression of Opa protein and downregulation of endogenous surface sialic acids analogous to what is seen for
N. gonorrhoeae
might be necessary for
N. meningitidis
as well.
M-Cell Surface β1 Integrin Expression and Invasin-Mediated Targeting of
Yersinia pseudotuberculosis
to Mouse Peyer’s Patch M Cells
Authors: Clark M. Ann , Hirst Barry H. , Jepson Mark A.
Published: December 2019

Quantitative analysis of
Yersinia pseudotuberculosis
infection of murine gut loops revealed that significantly more wild-type bacteria associated with Peyer’s patch M cells than with dome enterocytes or goblet cells. An invasin-deficient mutant was significantly attenuated for M-cell invasion, while β1 integrin expression was demonstrated in the apical membranes of M cells but not enterocytes. M-cell targeting by
Yersinia pseudotuberculosis
in vivo may, therefore, be mediated primarily by the interaction of invasin with cell surface β1 integrins.
Expression of Heat-Stable Enterotoxin STb by AdherentEscherichia coliIs Not Sufficient To Cause Severe Diarrhea in Neonatal Pigs
Authors: Casey Thomas A. , Herring Cheryl J. , Schneider Robert A. , Bosworth Brad T. , Whipp Shannon C.
Published: December 2019

The role ofEscherichia coliheat-stable enterotoxin B (STb) in neonatal porcine diarrhea caused by enterotoxigenicE. coliwas examined by comparing adherent isogenic strains with or without STb. The cloned STb gene (in the plasmid pRAS1) was electroporated into a nonenterotoxigenic strain (226M) which expresses the F41 adhesin. Strain 226M pRAS1 adhered and expressed STb in vivo, causing fluid secretion in ligated ileal loops in neonatal pigs. Although strain 226M pRAS1 caused very mild diarrhea in some orally inoculated neonatal pigs, the weight loss in these pigs was similar to that caused by the parental strain without STb. We conclude that STb does not significantly contribute to diarrhea caused by enterotoxigenicE. coliin neonatal pigs.
The
Haemophilus influenzae
HtrA Protein Is a Protective Antigen
Authors: Loosmore Sheena M. , Yang Yan-ping , Oomen Ray , Shortreed Jean M. , Coleman Debbie C. , Klein Michel H.
Published: December 2019

The
htrA
gene from two strains of nontypeable
Haemophilus influenzae
has been cloned and sequenced, and the encoded approximately 46-kDa HtrA proteins were found to be highly conserved.
H. influenzae
HtrA has approximately 55% identity with the
Escherichia coli
and
Salmonella typhimurium
HtrA stress response proteins, and expression of the
H. influenzae htrA
gene was inducible by high temperature. Recombinant HtrA (rHtrA) was expressed from
E. coli
, and the purified protein was found to have serine protease activity. rHtrA was found to be very immunogenic and partially protective in both the passive infant rat model of bacteremia and the active chinchilla model of otitis media. Immunoblot analysis indicated that HtrA is antigenically conserved in encapsulated and nontypeable
H. influenzae
species. Site-directed mutagenesis was performed on the
htrA
gene to ablate the endogenous serine protease activity of wild-type HtrA, and it was found that eight of nine recombinant mutant proteins had no measurable residual proteolytic activity. Two mutant proteins were tested in the animal protection models, and one, H91A, was found to be partially protective in both models. H91A HtrA may be a good candidate antigen for a vaccine against invasive
H. influenzae
type b disease and otitis media and is currently in phase I clinical trials.
Protein Deficiency Induces Alterations in the Distribution of T-Cell Subsets in Experimental Pulmonary Tuberculosis
Authors: Mainali Elsie S. , McMurray David N.
Published: December 2019

Previous research has suggested that dietary protein deficiency alters resistance to experimental pulmonary tuberculosis, in part, by affecting the distribution and trafficking of antigen-reactive T cells. In this study, guinea pigs were maintained on either a protein-deficient (10% ovalbumin) or control (30% ovalbumin) diet and infected 4 to 6 weeks later with a low dose of virulent
Mycobacterium tuberculosis
H37Rv by the respiratory route. Monoclonal antibodies directed against the CD4 or CD8 markers on guinea pig lymphocytes were used in a flow cytofluorometric assay to determine the proportion of each subset in the peripheral circulation, spleen, and bronchotracheal lymph nodes at 4 weeks after infection. In uninfected guinea pigs, only the spleen exhibited an effect of diet on T-cell distribution, with small but consistent reductions in the proportions of both CD4 and CD8 T lymphocytes. However, following infection, protein deficiency exerted a profound effect on T-cell distribution. Malnourished, tuberculous guinea pigs harbored only 20 and 60% of the T cells (as a proportion of total lymphoid cells) found in the spleen and blood, respectively, of their well-nourished counterparts. Normal relative proportions of CD4 and CD8 cells were observed, however. In striking contrast, the bronchotracheal lymph nodes of protein-deprived guinea pigs with tuberculosis contained more than twice the numbers of T cells of control guinea pigs, and the normal CD4-to-CD8 ratio was reversed. Peripheral T-cell function, as measured by the delayed hypersensitivity skin test to tuberculin, and antigen-induced lymphoproliferation in vitro were markedly suppressed in protein-malnourished animals. Conversely, purified protein derivative-induced (but not concanavalin A-induced) proliferation was significantly enhanced in cultures of lymph node cells from protein-deprived tuberculous animals. Taken together, these results suggest that immunological abnormalities and loss of antimycobacterial resistance in the lungs of protein-deficient guinea pigs may be explained, in part, by sequestration of antigen-reactive T cells in the lymph nodes draining the site of infection.
Mutation in the
peb1A
Locus of
Campylobacter jejuni
Reduces Interactions with Epithelial Cells and Intestinal Colonization of Mice
Authors: Pei Zhiheng , Burucoa Christophe , Grignon Bernadette , Baqar Shahida , Huang Xiao-Zhe , Kopecko Dennis J. , Bourgeois A. L. , Fauchere Jean-Louis , Blaser Martin J.
Published: December 2019

Campylobacter jejuni
is one of the leading causes of bacterial diarrhea throughout the world. We previously found that PEB1 is a homolog of cluster 3 binding proteins of bacterial ABC transporters and that a
C. jejuni
adhesin, cell-binding factor 1 (CBF1), if not identical to, contains PEB1. A single protein migrating at approximately 27 to 28 kDa was recognized by anti-CBF1 and anti-PEB1. To determine the role that the operon encoding PEB1 plays in
C. jejuni
adherence,
peb1A
, the gene encoding PEB1, was disrupted in strain 81-176 by insertion of a kanamycin resistance gene through homologous recombination. Inactivation of this operon completely abolished expression of CBF1, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. In comparison to the wild-type strain, the mutant strain showed 50- to 100-fold less adherence to and 15-fold less invasion of epithelial cells in culture. Mouse challenge studies showed that the rate and duration of intestinal colonization by the mutant were significantly lower and shorter than with the wild-type strain. In summary, PEB1 is identical to a previously identified cell-binding factor, CBF1, in
C. jejuni
, and the
peb1A
locus plays an important role in epithelial cell interactions and in intestinal colonization in a mouse model.
Curli, Fibrous Surface Proteins ofEscherichia coli, Interact with Major Histocompatibility Complex Class I Molecules
Authors: Olsén Arne , Wick Mary Jo , Mörgelin Matthias , Björck Lars
Published: December 2019

Curli are thin, coiled fibers expressed on the surface ofEscherichia colithat bind several matrix and plasma proteins such as fibronectin, laminin, plasminogen, tissue plasminogen activator, and H-kininogen. In this work, we examined the interactions between curli-expressingE. coliand human major histocompatibility complex class I (MHC-I) and class II (MHC-II) molecules. CurliatedE. coliwas found to interact with an MHC-I-expressing lymphoma cell line as shown by scanning electron microscopy, whereas the binding to a mutant variant of this cell line expressing small amounts of MHC-I molecules was significantly lower. Moreover, curli-expressingE. colibound purified radiolabeled MHC-I but not MHC-II molecules, whereas an isogenic curli-deficient mutant strain showed no affinity for either MHC-I or MHC-II. Purified insoluble curli could also bind125I-labeled MHC-I molecules, and in Western blot experiments the 15-kDa curlin subunit protein bound intact MHC-I molecules as well as β2-microglobulin, the light chain of MHC-I molecules. A direct interaction between monomeric MHC-I molecules and a bacterial surface protein has previously not been reported. The binding of curli to MHC-I molecules, which are present on virtually all cells in higher vertebrates, will provide curliatedE. coliwith ample opportunities to interact with a great variety of hosts and host cells. This should facilitate the adaptation ofE. colito different ecological niches, and in human infections the interaction between curli and MHC-I molecules could contribute to adherence and colonization.
Chlamydial Infection in Inducible Nitric Oxide Synthase Knockout Mice
Authors: Igietseme Joseph U. , Perry Linda L. , Ananaba Godwin A. , Uriri Ijindah M. , Ojior Omegbhai O. , Kumar Shantha N. , Caldwell Harlan D.
Published: December 2019

Type 1 CD4+-T-cell-mediated immunity is crucial for the resolution of chlamydial infection of the murine female genital tract. Previous studies demonstrating a correlation between CD4+-T-cell-mediated inhibition of chlamydial growth and gamma interferon (IFN-γ)-mediated induction of nitric oxide synthase suggested a potential role for the nitric oxide (NO) effector pathway in the clearance ofChlamydiafrom genital epithelial cells by the immune system. To clarify the role of this pathway, the growth levels ofChlamydia trachomatisorganisms in normal (iNOS+/+) mice and in genetically engineered mice lacking the inducible nitric oxide synthase (iNOS) gene (iNOS−/−mice) were compared. There was no significant difference in the course of genital chlamydial infections in iNOS+/+and iNOS−/−mice as determined by recovery ofChlamydiaorganisms shed from genital epithelial cells. Dissemination ofChlamydiato the spleen and lungs occurred to a greater extent in iNOS−/−than in iNOS+/+mice, which correlated with a marginal increase in the susceptibility of macrophages from iNOS−/−mice to chlamydial infection in vitro. However, infections were rapidly cleared from all affected tissues, with no clinical signs of disease. The finding of minimal dissemination in iNOS−/−mice suggested that activation of the iNOS effector pathway was not the primary target of IFN-γ during CD4+-T-cell-mediated control of chlamydial growth in macrophages because previous reports demonstrated extensive and often fatal dissemination ofChlamydiain mice lacking IFN-γ. In summary, these results indicate that the iNOS effector pathway is not required for elimination ofChlamydiafrom epithelial cells lining the female genital tract of mice although it may contribute to the control of dissemination ofC. trachomatisby infected macrophages.
Toxoplasma gondiiTriggers Granulocyte-Dependent Cytokine-Mediated Lethal Shock ind-Galactosamine-Sensitized Mice
Authors: Marshall Anthony J. , Denkers Eric Y.
Published: December 2019

To investigate the capacity ofToxoplasma gondiito induce cytokine-mediated toxicity, we employed a murine model of lethal shock in which hypersensitivity to microbial toxins is induced byd-galactosamine (d-Gal). Animals injected withd-Gal and tachyzoite lysate died within 12 to 24 h, whereas administration ofd-Gal or lysate alone was nonlethal. Analyses of plasma cytokines revealed peaks of tumor necrosis factor (TNF) alpha and interleukin-12 (IL-12) 1 and 3 to 5 h after injection, respectively, and gradually rising levels of gamma interferon (IFN-γ) continuing until death. Nitric oxide (NO) levels in serum paralleled IFN-γ production. Transaminase assays revealed elevated levels of liver-associated enzymes in sera of lethally injected mice, indicating severe hepatic damage. Depletion of IL-12, TNF, IFN-γ, and NO rescued mice from the lethal effect of antigen (Ag) andd-Gal. T-cell-deficient animals remained sensitive tod-Gal and lysate, suggesting that T lymphocytes do not contribute to the response. Nevertheless, monoclonal antibody (MAb)-mediated granulocyte depletion completely abrogatedd-Gal- and Ag-induced mortality and accompanying liver pathology. Finally, mice acutely infected withT. gondiidisplayed highly elevated NO and liver enzyme levels in serum immediately prior to death, and administration of anti-TNF MAb prolonged survival by approximately 24 h. Our results demonstrate thatT. gondiiinduces lethal inflammatory cytokine shock ind-Gal-sensitized animals and suggest that a similar pathology may contribute to manifestations of acute toxoplasmosis.
The N-Terminal Part of the Enzyme Component (C2I) of the Binary
Clostridium botulinum
C2 Toxin Interacts with the Binding Component C2II and Functions as a Carrier System for a Rho ADP-Ribosylating C3-Like Fusion Toxin
Authors: Barth H. , Hofmann F. , Olenik C. , Just I. , Aktories K.
Published: December 2019

The binary actin–ADP-ribosylating
Clostridium botulinum
C2 toxin consists of the enzyme component C2I and the binding component C2II, which are separate proteins. The active component C2I enters cells through C2II by receptor-mediated endocytosis and membrane translocation. The N-terminal part of C2I (C2IN), which consists of 225 amino acid residues but lacks ADP-ribosyltransferase activity, was identified as the C2II contact site. A fusion protein (C2IN-C3) of C2IN and the full-length C3-like ADP-ribosyltransferase from
Clostridium limosum
was constructed. The fusion protein C2IN-C3 ADP-ribosylated Rho but not actin in CHO cell lysates. Together with C2II, C2IN-C3 induced complete rounding up of CHO and HeLa cells after incubation for 3 h. No cell rounding was observed without C2II or with the original C3-like transferase from
C. limosum
. The data indicate that the N-terminal 225 amino acid residues of C2I are sufficient to cause the cellular uptake of
C. limosum
transferase via the binding component of C2II, thereby increasing the cytotoxicity of the C3-like exoenzyme several hundred-fold.
Defects in Type III Secretion Correlate with Internalization of
Pseudomonas aeruginosa
by Epithelial Cells
Authors: Hauser Alan R. , Fleiszig Suzanne , Kang Pil Jung , Mostov Keith , Engel Joanne N.
Published: December 2019

Previous characterization of
Pseudomonas aeruginosa
clinical isolates has demonstrated an inverse correlation between cytotoxicity and internalization by epithelial cells. To further investigate this relationship, we tested PA103, a cytotoxic
P. aeruginosa
strain, and 33 isogenic noncytotoxic transposon mutants for internalization by Madin-Darby canine kidney cells. The majority of the mutants were not internalized, demonstrating that an inverse correlation between cytotoxicity and bacterial uptake by epithelial cells is not absolute. Six of the noncytotoxic mutants, however, demonstrated measurable levels of internalization by standard aminoglycoside exclusion assays even though internalization of wild-type strain PA103 was not detectable. All six had evidence of protein secretion defects involving two proteins, a 40-kDa protein and a 32-kDa protein. These proteins, designated PepB (for
Pseudomonas
exoprotein B) and PepD, respectively, each had characteristics of type III transported proteins. In addition, nucleotide sequencing studies demonstrated that PepB and PepD are homologs of YopB and YopD, respectively, type III secreted proteins of
Yersinia
spp. necessary for the translocation of effector molecules into the cytoplasmic compartment of eukaryotic cells. Thus, while many mutations in PA103 result in loss of cytotoxicity without an appreciable increase in internalization, defects in transport of type III secretion proteins PepB and PepD correlate with both loss of cytotoxicity and gain of internalization. These results are consistent with type III secretion of an inhibitor of internalization that requires PepB and PepD for translocation into the host cell.
Molecular Analysis of Shiga Toxigenic
Escherichia coli
O111:H
−
Proteins Which React with Sera from Patients with Hemolytic-Uremic Syndrome
Authors: Voss Elena , Paton Adrienne W. , Manning Paul A. , Paton James C.
Published: December 2019

Western blot analysis was used to assess the reactivity of convalescent-phase sera from patients who were associated with an outbreak of hemolytic-uremic syndrome (HUS) caused by fermented sausage contaminated with Shiga toxin-producing
Escherichia coli
(STEC). The predominant STEC isolated from HUS patients belonged to serotype O111:H
−
, and reactivity to O111:H
−
whole-cell lysates, treated or untreated with proteinase K, was examined. As expected, all five serum samples demonstrated a marked anti-lipopolysaccharide response, but several protein bands were also immunoreactive, particularly one with an apparent size of 94 kDa. One convalescent-phase serum sample was subsequently used to screen an O111:H
−
cosmid bank and 2 of 900 cosmid clones were found to be positive, both of which contained a similar DNA insert. Western blot analysis of one of these clones identified three major immunoreactive protein bands of approximately 94, 70, and 50 kDa. An immune response to the three proteins was detectable with all five convalescent-phase serum samples but not with healthy human serum. Immunoreactive 94- and 50-kDa species were produced by a deletion derivative of the cosmid containing a 7-kb STEC DNA insert. Sequence analysis of this region indicated that it is part of the locus for enterocyte effacement, including the
eaeA
gene which encodes intimin. The deduced amino acid sequence of the O111:H
−
intimin was 88.6% identical to intimin from O157:H7 STEC, and the most divergent region was the 200 residues at the carboxyl terminus, which were only 75% identical. Such variation may be antigenically significant as serum from a HUS patient infected only with the O111:H
−
STEC reacted with intimin from an enteropathogenic
E. coli
O111 strain, as well as several other
eaeA
-positive STEC isolates, but not with an
eaeA
-positive STEC belonging to serotype O157:H
−
. Sera from two of the other HUS patients also failed to react with intimin from this latter strain. However, intimin from O157:H
−
STEC did react with serum from a patient infected with both O111:H
−
and O157:H
−
STEC.