bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of Arg-Gingipain A in Virulence of
Porphyromonas gingivalis
Authors: Tokuda Masayuki , Karunakaran Thonthi , Duncan Margaret , Hamada Nobushiro , Kuramitsu Howard
Published: December 2019

In order to access the role of the
Porphyromonas gingivalis
Arg-gingipain proteases in the virulence of this organism, a mutant defective in the
rgpA
gene was constructed in strain 381. This mutant, MT10, displayed only 40% of the Arg-specific cysteine protease activity of the wild-type strain. In addition, MT10, as well as the recently characterized protease mutant G-102, which is defective in the
rgpB
gene, displayed reduced self-aggregation, hemagglutination, and the ability to bind to immobilized type I collagen compared to levels of the wild-type parent. However, unlike mutant G-102, the
rgpA
mutant displayed increased binding to epithelial cells relative to that of the parental organism. Mutant MT10 also did not express detectable levels of the FimA protein as assessed by both Western and Northern blotting or fimbriae visible by electron microscopy of the cells. Furthermore, the ability of MT10 to degrade rat tail collagen fibers when it was cultured at 37°C was markedly attenuated compared to that of strain 381. These results suggest that Arg-gingipain A may play a significant role in the pathogenicity of
P. gingivalis
by altering the colonization and toxic properties of the organism.
Human T-Cell Responses after Vaccination with the Norwegian Group B Meningococcal Outer Membrane Vesicle Vaccine
Authors: Næss Lisbeth Meyer , Oftung Fredrik , Aase Audun , Wetzler Lee M. , Sandin Randi , Michaelsen Terje E.
Published: December 2019

We have analyzed human T-cell responses in parallel with serum immunoglobulin G (IgG) antibody levels after systemic vaccination with the Norwegian group BNeisseria meningitidisouter membrane vesicle (OMV) vaccine. Ten adult volunteers, with no or very low levels of serum IgG antibodies against meningococci, received three doses intramuscularly of the OMV vaccine (at weeks 0, 6, and 46). T-cell proliferation against the OMV vaccine, purified outer membrane proteins (PorA and PorB), and control antigens (Mycobacterium bovisBCG vaccine and tetanus toxoid) was measured by thymidine incorporation of peripheral blood mononuclear cells before and after vaccination. The results showed that vaccination with OMV elicits strong primary and booster T-cell responses specific to OMV as well as the PorA (class 1) protein and significant, but markedly lower, responses against the PorB (class 3) protein. The median responses to OMV and PorA were 26 and 16 times the prevaccination levels, respectively. Most of the vaccinees showed low T-cell responses against OMV and PorA before vaccination, and the maximum T-cell responses to all vaccine antigens were usually obtained after the second vaccine dose. We found a positive correlation between T-cell responses and anti-OMV IgG antibody levels (r= 0.50,P< 0.0001, for OMV and PorA). In addition, we observed a progressive increase in the percentage of CD45R0+(memory) CD4-positive T cells (P= 0.002). In conclusion, we have shown that the Norwegian OMV vaccine against meningococcal B disease induced antigen-specific T-cell responses, kinetically accompanied by serum IgG responses, and that vaccination increased the proportion of memory T-helper cells.
Immunoglobulins to Group A Streptococcal Surface Molecules Decrease Adherence to and Invasion of Human Pharyngeal Cells
Authors: Fluckiger U. , Jones K. F. , Fischetti V. A.
Published: December 2019

The M protein is one of the most important virulence factors of group A streptococci (Streptococcus pyogenes) and may play an important role in the first steps of streptococcal infection. Since acute pharyngitis is a frequently occurring infectious disease caused by these bacteria, we wished to know whether antibodies to the M protein or other surface components inhibit adherence and internalization of streptococci to pharyngeal cells. We investigated the role of whole human secretory immunoglobulin A (sIgA), M6 protein-specific sIgA, and M6 protein-specific serum IgG in the inhibition of streptococcal adherence and internalization to cultured human pharyngeal cells.S. pyogenesD471, which produces a type 6 M protein (M+), and its isogenic M-negative (M−) derivative JRS75 were tested. Purified whole sIgA, M protein-specific sIgA, and sIgA preabsorbed with M protein were able to decrease significantly the adherence of streptococci to pharyngeal cells. Purified IgG against the M6 protein did not diminish the attachment of streptococci to the pharyngeal cells but did reduce internalization. Thus, our data suggest that secretory IgA may play a key role in preventing streptococcal infection at mucosal surfaces by blocking adherence while affinity-purified anti-M protein-specific IgG blocks epitopes responsible for invasion.
An Essential Role for Gamma Interferon in Innate Resistance toShigella flexneriInfection
Authors: Way Sing Sing , Borczuk Alain C. , Dominitz Rene , Goldberg Marcia B.
Published: December 2019

Shigellaspp. are the major cause of bacillary dysentery worldwide. To identify immune effectors associated with protection of the naive host during infection, the susceptibility to pulmonaryShigellainfection of each of various mouse strains that have a targeted deletion in a specific aspect of the immune system was evaluated. Our results demonstrate that mice deficient in gamma interferon are 5 orders of magnitude more susceptible toShigellathan are wild-type mice, whereas mice deficient in B and T lymphocytes or in T lymphocytes alone exhibit no difference in susceptibility. Significantly lower numbers of shigellae were recovered from immunocompetent compared with gamma-interferon-deficient mice after infection. While immunocompetent mice were able to clear a sublethalShigellainoculum by day 5 postinfection, progressively increasing numbers of shigellae were cultured from the lungs of gamma interferon-deficient mice over the same period. Histopathology of the lungs from immunocompetent mice infected with a sublethalShigellainoculum showed mild inflammatory changes, whereas the lungs from gamma interferon-deficient mice demonstrated progressively worsening acute bronchiolitis with ulceration. Further, the time to death in gamma interferon-deficient mice correlates inversely with the size of theShigellainoculum. To identify the cellular source of gamma interferon, we infected SCID mice, T-cell-receptor-deficient mice, beige mice (a mouse strain deficient in natural killer [NK] cell activity), and mice depleted of NK cells using anti-asialo-GM1. Each NK cell-deficient mouse strain exhibited a 10-fold-greater susceptibility toShigellainfection than immunocompetent mice. To test the protective effects of gamma interferon in vitro, survival of intracellularShigellawas examined in primary macrophages from wild-type mice, primary macrophages from gamma interferon-deficient mice, a macrophage cell line, and a fibroblast cell line. Following activation with gamma interferon, each cell type eradicated intracellularShigella, while nonactivated macrophages fosteredShigellareplication and nonactivated fibroblast cells fostered bothShigellareplication and intercellular spread. Taken together, these data establish that NK cell-mediated gamma interferon is essential to resistance following primaryShigellainfection.
A New Member of the S-Layer Protein Family: Characterization of the
crs
Gene from
Campylobacter rectus
Authors: Wang Beinan , Kraig Ellen , Kolodrubetz David
Published: December 2019

Strains of the periodontal pathogen
Campylobacter rectus
express a 150- to 166-kDa protein on their cell surface. This protein forms a paracrystalline lattice, called the surface layer (S-layer), on the outer membrane of this gram-negative bacterium. To initiate a genetic analysis of the function of the S-layer in the pathogenesis of
C. rectus
, we have cloned and characterized its gene. The S-layer gene (
crs
) from
C. rectus
314 encodes a cell surface protein which does not have a cleaved signal peptide at its amino terminus. Although the amino acid sequence deduced from the
crs
gene has 50% identity with the amino-terminal 30 amino acids of the four S-layer proteins from
Campylobacter fetus
, the similarity decreases to less than 16% over the rest of the protein. Thus, the
crs
gene from
C. rectus
encodes a novel S-layer protein whose precise role in pathogenesis may differ from that of S-layer proteins from other organisms. Southern and Northern blot analyses with probes from different segments of the
crs
gene indicate that the S-layer gene is a single-copy, monocistronic gene in
C. rectus
. RNA end mapping and sequence analyses were used to define the
crs
promoter; there is an exact match to the
Escherichia coli
−10 promoter consensus sequence but only a weak match to the −35 consensus element. Southern blots of DNA from another strain of
C. rectus
, ATCC 33238, demonstrated that the
crs
gene is also present in that strain but that there are numerous restriction fragment length polymorphisms in the second half of the gene. This finding suggests that the carboxy halves of the S-layer proteins from strains 314 and 33238 differ. It remains to be determined whether the diversities in sequence are reflected in functional or antigenic differences important for the pathogenesis of different
C. rectus
isolates.
Comparison of an Adherence Domain and a Structural Region ofStreptococcus mutansAntigen I/II in Protective Immunity against Dental Caries in Rats after Intranasal Immunization
Authors: Hajishengallis George , Russell Michael W. , Michalek Suzanne M.
Published: December 2019

Previous studies have identified an N-terminal saliva-binding region (SBR) onStreptococcus mutanssurface antigen I/II (AgI/II) and suggested its importance in the initial adherence ofS. mutansto saliva-coated tooth surfaces and subsequent development of dental caries. In this study, we compared the SBR with a C-terminal structural region of AgI/II (AgII) in their abilities to induce protective immunity against caries in rats. When SBR, AgII, or the whole AgI/II molecule was administered intranasally as a conjugate with the B subunit of cholera toxin (CT), in the presence of CT adjuvant, substantial levels of salivary immunoglobulin A anti-AgI/II antibodies were induced. Evaluation of caries activity showed that the SBR, though not as protective as the parent molecule, was superior to AgII and thus can be further considered as a component in a multivalent caries vaccine.
Cytokine Induction in Murine Macrophages Infected with Virulent and Avirulent
Rhodococcus equi
Authors: Giguère Steeve , Prescott John F.
Published: December 2019

To look for a possible correlation between the virulence of
Rhodococcus equi
and its cytokine-inducing capacity, we evaluated intracellular survival and measured cytokine induction by mouse macrophages infected with a virulent strain containing an 85-kb plasmid and expressing VapA (103
+
), its avirulent plasmid-cured derivative (103
−
), and heat-killed 103
+
(HK). After incubation with similar numbers of bacteria, macrophages infected with 103
−
contained significantly more organisms than those infected with 103
+
or HK. The number of bacteria in the macrophages infected with 103
−
and HK decreased progressively, whereas the 103
+
numbers remained constant over 48 h. Interleukin 1β (IL-1β), IL-6, IL-10, IL-12 p40, and tumor necrosis factor alpha (TNF-α) mRNA induction peaked at 4 h and returned to baseline between 12 and 48 h postinfection. IL-1β, IL-6, IL-10, and TNF-α concentrations assessed by enzyme-linked immunosorbent assay generally agreed well with mRNA expression; IL-12 could, however, not be detected. For all the cytokines detected, mean concentrations in the supernatants were consistently higher in the 103
−
-infected monolayers than in those infected with 103
+
, although, with the exception of IL-1β, the differences were not statistically significant.
R. equi
HK was a poor inducer of cytokine production. In conclusion, virulent and avirulent
R. equi
strains induced similar levels of cytokine synthesis. The slightly greater induction of most cytokines observed following infection with 103
−
is likely secondary to greater uptake by macrophages rather than to a direct role of VapA or another plasmid-encoded product in downregulating cytokine induction.
Pasteurella haemolyticaLeukotoxin-Induced Increase in Phospholipase A2Activity in Bovine Neutrophils
Authors: Wang Zuncai , Clarke Cyril , Clinkenbeard Kenneth
Published: December 2019

Exposure of bovine neutrophils toPasteurella haemolyticaleukotoxin (LKT) stimulates the production of leukotriene B4(LTB4), which is believed to be an important chemotactic agent in the development of acute fibrinopurulent pneumonic infection in cattle. The involvement of phospholipase A2(PLA2) in LKT-induced synthesis of LTB4was studied by using bovine neutrophils labeled with3H-arachidonate ([3H]AA). Incubation of isolated neutrophils with [3H]AA resulted in incorporation of radioactivity in the PLA2substrates phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine. Exposure of radiolabeled neutrophils to LKT caused concentration- and time-dependent release of radioactivity and redistribution of radioactivity in neutrophil membranes consistent with utilization of phosphoglyceride substrate and release of free fatty acid and eicosanoid products. These LKT-induced effects could be inhibited by pretreatment with arachidonyl trifluoromethyl ketone, an inhibitor of type IV cytoplasmic PLA2, and were dependent on extracellular calcium. These results support the conclusion that LKT-induced synthesis of LTB4involves a calcium-mediated increase in PLA2activity.
Involvement of T Cells in Enhanced Resistance toKlebsiella pneumoniaeSepticemia in Mice Treated with Liposome-Encapsulated Muramyl Tripeptide Phosphatidylethanolamine or Gamma Interferon
Authors: ten Hagen Timo L. M. , van Vianen Wim , Savelkoul Huub F. J. , Heremans Hubertine , Buurman Wim A. , Bakker-Woudenberg Irma A. J. M.
Published: December 2019

We have previously shown that prophylactic administration of the liposome-encapsulated immunomodulating agents muramyl tripeptide phosphatidylethanolamine (MTPPE) and gamma interferon (IFN-γ) results in strongly increased survival of mice from a normally lethal septicemia withKlebsiella pneumoniae. It was anticipated that the treatment acts on macrophages and nonspecifically augments host resistance to various infections. In the present study, we provide evidence for a key role for T cells in host defense potentiation by the liposomal immunomodulators towardK. pneumoniaesepticemia. It is shown that both CD4 and CD8 cells are important in immunomodulation, most likely due to production of IFN-γ. Depletion of circulating IFN-γ resulted in strong reduction of the antimicrobial host defense activation. Administration of interleukin-10 resulted in decreased antimicrobial host defense activation by liposomal immunomodulators. Moreover, administration of liposomal immunomodulators was shown to induce predominantly T-helper type 1 (Th1) cell populations in the spleen. These findings indicate that immunomodulation with liposomal MTPPE and IFN-γ favors Th1 and NK cell activation.
Burkholderia cepacia
Produces a Hemolysin That Is Capable of Inducing Apoptosis and Degranulation of Mammalian Phagocytes
Authors: Hutchison Michael L. , Poxton Ian R. , Govan John R. W.
Published: December 2019

Burkholderia cepacia
is an opportunistic pathogen that has become a major threat to individuals with cystic fibrosis (CF). In approximately 20% of patients, pulmonary colonization with
B. cepacia
leads to cepacia syndrome, a fatal fulminating pneumonia sometimes associated with septicemia. It has been reported that culture filtrates of clinically derived strains of
B. cepacia
are hemolytic. In this study, we have characterized a factor which contributes to this hemolytic activity and is secreted from
B. cepacia
J2315, a representative of the virulent and highly transmissible strain belonging to the recently described genomovar III grouping. Biochemical data from the described purification method for this hemolysin allows us to hypothesize that the toxin is a lipopeptide. As demonstrated for other lipopeptide toxins, the hemolysin from
B. cepacia
was surface active and lowered the surface tension of high-pressure liquid chromatography-grade water from 72.96 to 29.8 mN m
−1
. Similar to reports for other pore-forming cytotoxins, low concentrations of the hemolysin were able to induce nucleosomal degradation consistent with apoptosis in human neutrophils and the mouse-derived macrophage-type cell line J774.2. Exposure of human neutrophils to higher concentrations of toxin resulted in increased activities of the neutrophil degranulation markers cathepsin G and elastase. Based on the results obtained in this study, we suggest a role that allows
B. cepacia
to thwart the immune response and a model of the events that may contribute to the severe inflammatory response in the lungs of CF patients.
Characterization of the Roles of Hemolysin and Other Toxins in Enteropathy Caused by Alpha-Hemolytic
Escherichia coli
Linked to Human Diarrhea
Authors: Elliott Simon J. , Srinivas S. , Albert M. John , Alam Khorshed , Robins-Browne Roy M. , Gunzburg Stuart T. , Mee Brian J. , Chang Barbara J.
Published: December 2019

Escherichia coli
strains producing alpha-hemolysin have been associated with diarrhea in several studies, but it has not been clearly demonstrated that these strains are enteropathogens or that alpha-hemolysin is an enteric virulence factor. Such strains are generally regarded as avirulent commensals. We examined a collection of diarrhea-associated hemolytic
E. coli
(DHEC) strains for virulence factors. No strain produced classic enterotoxins, but they all produced an alpha-hemolysin that was indistinguishable from that of uropathogenic
E. coli
strains. DHEC strains also produced other toxins including cytotoxic necrotizing factor 1 (CNF1) and novel toxins, including a cell-detaching cytotoxin and a toxin that causes HeLa cell elongation. DHEC strains were enteropathogenic in the RITARD (reversible intestinal tie adult rabbit diarrhea) model of diarrhea, causing characteristic enteropathies, including inflammation, necrosis, and colonic cell hyperplasia in both small and large intestines. Alpha-hemolysin appeared to be a major virulence factor in this model since it conferred virulence to nonpathogenic
E. coli
strains. Other virulence factors also appear to be contributing to virulence. These findings support the epidemiologic link to diarrhea and suggest that further research into the role of DHEC and alpha-hemolysin in enteric disease is warranted.
Opsonic Antibodies to the Surface M Protein of Group A Streptococci in Pooled Normal Immunoglobulins (IVIG): Potential Impact on the Clinical Efficacy of IVIG Therapy for Severe Invasive Group A Streptococcal Infections
Authors: Basma Hesham , Norrby-Teglund Anna , McGeer Allison , Low Donald E. , El-Ahmedy Omar , Dale James B. , Schwartz Benjamin , Kotb Malak
Published: December 2019

The surface M protein of group A streptococci (GAS) is one of the major virulence factors for this pathogen. Antibodies to the M protein can facilitate opsonophagocytosis by phagocytic cells present in human blood. We investigated whether pooled normal immunoglobulin G (IVIG) contains antibodies that can opsonize and enhance the phagocytosis of type M1 strains of GAS and whether the levels of these antibodies vary for different IVIG preparations. We focused on the presence of anti-M1 antibodies because the M1T1 serotype accounts for the majority of recent invasive GAS clinical isolates in our surveillance studies. The level of anti-M1 antibodies in three commercial IVIG preparations was determined by enzyme-linked immunosorbent assay (ELISA), and the opsonic activity of these antibodies was determined by neutrophil-mediated opsonophagocytosis of a representative M1T1 isolate. High levels of opsonic anti-M1 antibodies were found in all IVIG preparations tested, and there was a good correlation between ELISA titers and opsonophagocytic activity. However, there was no significant difference in the levels of opsonic anti-M1 antibodies among the various IVIG preparations or lots tested. Adsorption of IVIG with M1T1 bacteria removed the anti-M1 opsonic activity, while the level of anti-M3 opsonophagocytosis was unchanged. Plasma was obtained from seven patients with streptococcal toxic shock syndrome who received IVIG therapy, and the level of anti-M1 antibodies was assessed before and after IVIG administration. A significant increase in the level of type M1-specific antibodies was found in the plasma of all patients who received IVIG therapy (P< 0.006). The results reveal another potential mechanism by which IVIG can ameliorate severe invasive group A streptococcal infections.
Mitogenic Response of Murine B Lymphocytes toSalmonella typhimuriumLipopolysaccharide Requires Protein Kinase C-Dependent Late Tyrosine Phosphorylations
Authors: Mey Anne , Revillard Jean-Pierre
Published: December 2019

Unlike the cross-linking of membrane immunoglobulins, the activation of B cells by lipopolysaccharide (LPS) does not involve the phosphoinositol turnover and the initial activation of tyrosine kinases. However, LPS-induced B-cell proliferation was inhibited by the tyrosine kinase inhibitors genistein and herbimycin A even when added 48 h after the beginning of the culture. Tyrosyl-phosphorylated proteins were detected by Western blotting after 24 h of culture with LPS, reaching a maximum concentration after 72 h. Late tyrosine phosphorylations were also detected in B cells activated for 72 h with anti-immunoglobulin M antibody and were abrogated by the protein synthesis inhibitor cycloheximide, the tyrosine kinase inhibitors genistein and herbimycin A, and the protein kinase C inhibitor chelerythrine. The role of protein kinase C in late tyrosine kinase activation is independent of Ca2+mobilization and was confirmed by detection of a comparable but restricted pattern of tyrosine-phosphorylated substrates in B cells treated with phorbol myristate acetate alone or in association with ionomycin. Tyrosine kinase activation was dependent on de novo protein synthesis. However, culture supernatants of LPS-activated B cells were devoid of mitogenic activity and induced a phosphorylation pattern more restricted than that achieved by LPS. Altogether these data indicate that proliferation signals induced by LPS or by the cross-linking of membrane immunoglobulins are controlled by late tyrosine phosphorylations occurring throughout the first 3 days of culture, controlled in part by protein kinase C activation, and dependent on the synthesis of an intermediate protein(s) either not secreted in the culture supernatant or present but biologically inactive in naive B cells.
Lipopolysaccharide and Its Analog Antagonists Display Differential Serum Factor Dependencies for Induction of Cytokine Genes in Murine Macrophages
Authors: Perera Pin-Yu , Qureshi Nilofer , Christ William J. , Stütz Peter , Vogel Stefanie N.
Published: December 2019

Monocytes/macrophages play a central role in mediating the effects of lipopolysaccharide (LPS) derived from gram-negative bacteria by the production of proinflammatory mediators. Recently, it was shown that the expression of cytokine genes for tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), and interferon-inducible protein-10 (IP-10) by murine macrophages in response to low concentrations of LPS is entirely CD14 dependent. In this report, we show that murine macrophages respond to low concentrations of LPS (≤2 ng/ml) in the complete absence of serum, leading to the induction of TNF-α and IL-1β genes. In contrast to the TNF-α and IL-1β genes, the IP-10 gene is poorly induced in the absence of serum. The addition of recombinant human soluble CD14 (rsCD14) had very little effect on the levels of serum-free, LPS-induced TNF-α, IL-1β, and IP-10 genes. In contrast, the addition of recombinant human LPS-binding protein (rLBP) had opposing effects on the LPS-induced TNF-α or IL-1β and IP-10 genes. rLBP inhibited LPS-induced TNF-α and IL-1β genes, while it reconstituted IP-10 gene expression to levels induced in the presence of serum. These results provide further evidence that the induction of TNF-α or IL-1β genes occurs via a pathway that is distinct from one that leads to the induction of the IP-10 gene and that the pathways diverge at the level of the initial interaction between LPS and cellular CD14. Additionally, the results presented here indicate that LPS structural analog antagonistsRhodobacter sphaeroidesdiphosphoryl lipid A and SDZ 880.431 are able to inhibit LPS-induced TNF-α and IL-1β in the absence of serum, while a synthetic analog ofRhodobacter capsulatuslipid A (B 975) requires both rsCD14 and rLBP to function as an inhibitor.
Modification of Ras in Eukaryotic Cells by
Pseudomonas aeruginosa
Exoenzyme S
Authors: McGuffie Eileen M. , Frank Dara W. , Vincent Timothy S. , Olson Joan C.
Published: December 2019

Genetic and functional data suggest that
Pseudomonas aeruginosa
exoenzyme S (ExoS), an ADP-ribosyltransferase, is translocated into eukaryotic cells by a bacterial type III secretory mechanism activated by contact between bacteria and host cells. Although purified ExoS is not toxic to eukaryotic cells, ExoS-producing bacteria cause reduced proliferation and viability, possibly mediated by bacterially translocated ExoS. To investigate the activity of translocated ExoS, we examined in vivo modification of Ras, a preferred in vitro substrate. The ExoS-producing strain
P. aeruginosa
388 and an isogenic mutant strain, 388Δ
exoS
, which fails to produce ExoS, were cocultured with HT29 colon carcinoma cells. Ras was found to be ADP-ribosylated during coculture with 388 but not with 388Δ
exoS
, and Ras modification by 388 corresponded with reduction in HT29 cell DNA synthesis. Active translocation by bacteria was found to be required, since exogenous ExoS, alone or in the presence of 388Δ
exoS
, was unable to modify intracellular Ras. Other ExoS-producing strains caused modification of Ras, indicating that this is not a strain-specific event. ADP-ribosylation of Rap1, an additional Ras family substrate for ExoS in vitro, was not detectable in vivo under conditions sufficient for Ras modification, suggesting possible ExoS substrate preference among Ras-related proteins. These results confirm that intracellular Ras is modified by bacterially translocated ExoS and that the inhibition of target cell proliferation correlates with the efficiency of Ras modification.
Clostridium difficileToxin B Induces Apoptosis in Intestinal Cultured Cells
Authors: Fiorentini Carla , Fabbri Alessia , Falzano Loredana , Fattorossi Andrea , Matarrese Paola , Rivabene Roberto , Donelli Gianfranco
Published: December 2019

Toxigenic strains of the anaerobic bacteriumClostridium difficileproduce at least two large, single-chain protein exotoxins involved in the pathogenesis of antibiotic-associated diarrhea and colitis. Toxin A (CdA) is a cytotoxic enterotoxin, while toxin B (CdB) is a more potent cytotoxin lacking enterotoxic activity. This study dealt with CdB, providing the first evidence that intestinal cells exposed to this toxin exhibit typical features of apoptosis in that a significant proportion of the treated cells displayed nuclear fragmentation and chromatin condensation. In keeping with ultrastructural data, CdB-treated cells showed the typical flow cytometric hallmark of apoptosis consisting of a distinct sub-G1peak. The CdB-induced apoptotic response was dose and time dependent and not simply due to the actin-disrupting effect of the toxin or to the subsequent impairment of cell anchorage. Rather, the inhibition of proteins belonging to the Rho family due to CdB seems to play a role in the induction of apoptosis in intestinal cells. The origin of cells and the growth rate may also be cofactors relevant to such a response.
Secretory Phospholipase A
2
Is the Principal Bactericide for Staphylococci and Other Gram-Positive Bacteria in Human Tears
Authors: Qu Xiao-Dan , Lehrer Robert I.
Published: December 2019

We examined human tears for molecules that killed gram-positive bacteria. The principal mediator of bactericidal activity against staphylococci proved to be a calcium-dependent enzyme, secretory phospholipase A
2
. Whereas the concentration of secretory phospholipase A
2
in the normal tear film exceeded 30 μg/ml, only 1.1 ng (<0.1 nM) of the enzyme per ml sufficed to kill
Listeria monocytogenes
and 15 to 80 ng/ml killed
Staphylococcus aureus
. Despite its efficacy against gram-positive bacteria, secretory phospholipase A
2
lacked bactericidal activity against gram-negative organisms (
Escherichia coli
,
Salmonella typhimurium
, and
Pseudomonas aeruginosa
) when tested in the ionic environment of tears. Given the presence of secretory phospholipase A
2
in tears, intestinal secretions, and leukocytes, this enzyme may play a substantial role in innate mucosal and systemic bactericidal defenses against gram-positive bacteria.
Time Course and Host Responses toEscherichia coliUrinary Tract Infection in Genetically Distinct Mouse Strains
Authors: Hopkins Walter J. , Gendron-Fitzpatrick Annette , Balish Edward , Uehling David T.
Published: December 2019

Recurrent urinary tract infections (UTIs) are a significant clinical problem for many women; however, host susceptibility factors have not been completely defined. The mouse model of induced UTI provides an experimental environment in which to identify specific host characteristics that are important in initial bacterial colonization of the urinary tract and in resolution of an infection. This study examined initial susceptibility, bacterial clearance, and host defense mechanisms during induction and resolution ofEscherichia coliUTIs in genetically distinct strains of mice. Of the ten inbred strains tested, six (BALB/c, C3H/HeN, C57BL/6, DBA.1, DBA.2, and AKR) showed progressive resolution of bladder infections over a 14-day period. A constant, low-level bladder infection was observed in SWR and SJL mice. High bladder infection levels persisted over the 14-day study period in C3H/HeJ and C3H/OuJ mice. Kidney infection levels generally correlated with bladder infection levels, especially in C3H/HeJ and C3H/OuJ mice, the two most susceptible strains, in which infections became more severe with time after challenge. The degree of inflammation in bladder and kidneys, as well as antibody-forming cell responses, positively correlated with infection intensity in all strains except C3H/HeJ, which had minimal inflammation despite high infection levels. These results demonstrate two important aspects of host defense against UTI. First, the innate immune response to an infection in the bladder or kidneys consists primarily of local inflammation, which is followed by an adaptive response characterized in part by an antibody response to the infecting bacteria. Second, a UTI will be spontaneously resolved in most cases; however, in mice with specific genetic backgrounds, a UTI can persist for an extended length of time. The latter result strongly suggests that the presence or absence of specific host genes will determine how effectively anE. coliUTI will be resolved.
The ImmunodominantBrugia malayiParamyosin as a Marker of Current Infection withWuchereria bancroftiAdult Worms
Authors: Langy Sandra , Plichart Catherine , Luquiaud Patrick , Williams Steven A. , Nicolas Luc
Published: December 2019

The full-length cDNA sequence encodingBrugia malayiL3 paramyosin has been isolated by immunoscreening a cDNA library with a mouse antiserum raised againstWuchereria bancroftiL3 infective larvae. A recombinant truncated form of paramyosin was expressed as a glutathioneS-transferase fusion protein and used to evaluate humoral responses of adults from aW. bancrofti-endemic area in French Polynesia according to their parasitological status. Immunoglobulin G4 (IgG4) preferentially bound to paramyosin inW. bancrofti-parasitized individuals, in contrast to unparasitized individuals, who harbored neither microfilaria nor Og4C3 adult worm circulating antigen. Reduction of the anti-paramyosin IgG4 titer following combined chemotherapy with diethylcarbamazine and ivermectin was significantly correlated with a reduction in the adult worm burden. This indicates that the presence of paramyosin-reactive IgG4 is associated with the presence of parasites and that reduction can be used as an immunological marker forW. bancrofticlearance.
Characterization of the Type 3 Fimbrial Adhesins of
Klebsiella
Strains
Authors: Sebghati Tricia A. Schurtz , Korhonen Timo K. , Hornick Douglas B. , Clegg Steven
Published: December 2019

The
Klebsiella pneumoniae
fimbrial adhesin, MrkD, mediates adherence to the basolateral surfaces of renal and pulmonary epithelia and to the basement membranes of tissues. Although all isolates possessing the MrkD adhesin mediate the agglutination, in vitro, of erythrocytes treated with tannic acid, the
mrkD
gene is not conserved within species. The ability of a plasmid-borne
mrkD
gene product to mediate binding to type V collagen is associated frequently with strains of
K. oxytoca
and rarely with strains of
K. pneumoniae
. In
K. pneumoniae
, the MrkD adhesin is located within a chromosomally borne gene cluster and mediates binding to collagen types IV and V. The plasmid-borne determinant,
mrkD
1P
, and the chromosomally borne gene,
mrkD
1C
, are not genetically related. Some strains of enterobacteria possess a
mrkD
1C
allele that is associated with hemagglutinating activity but does not bind to either type IV or type V collagen.
Physical Limitations onSalmonella typhiEntry into Cultured Human Intestinal Epithelial Cells
Authors: Huang Xiao-Zhe , Tall Ben , Schwan William R. , Kopecko Dennis J.
Published: December 2019

Kinetic studies ofSalmonella typhiinvasion of INT407 cells at different multiplicities of infection (MOIs) have revealed a strict physical limitation onS. typhientry at MOIs of ≥40. Staining of infected monolayers to distinguish intracellular from extracellular bacteria revealed that all monolayer cells are susceptible to infection and that internalized bacteria are typically contained in one to three separate clusters per cell during the first 60 min. Scanning and transmission electron microscopic analyses of time course-infected monolayers showed that at early times postinfection, bacteria bind to shortened, coalesced microvilli in one to three focal aggregate structures per host cell surface. As reported previously forS. typhimurium, focal aggregates progress to conical membrane ruffles that appear to engulf one or a few centrally containedS. typhicells by a macropinocytic process, which enhanced the entry of simultaneously addedEscherichia coliHB101 about 30-fold. Additionally, kinetic studies showed that at an MOI of ≃400, maximalS. typhientry is virtually completed within 30 to 35 min. Monolayers pretreated withS. typhifor 30 min to saturate the entry process were severely reduced in the ability to internalize subsequently added kanamycin-resistant strains ofS. typhiorS. typhimurium, butE. coliHB101(pRI203) expressing the clonedYersinia invgene was not reduced in entry. In invasion inhibition assays, anti-β1 integrin antibodies markedly reducedE. coliHB101(pRI203) invasion efficiency but did not reduceS. typhientry. Collectively, these data provide direct physical and visual evidence which indicates thatS. typhiorganisms are internalized at a limited number (i.e., two to four) of sites on host cells.S. typhiandS. typhimuriumlikely share INT407 cell entry receptors which do not appear to be members of the β1 integrin superfamily.
Protective Roles of γδ T Cells and Interleukin-15 inEscherichia coliInfection in Mice
Authors: Takano M. , Nishimura H. , Kimura Y. , Mokuno Y. , Washizu J. , Itohara S. , Nimura Y. , Yoshikai Y.
Published: December 2019

The number of γδ T cells in the peritoneal cavity was increased after an intraperitoneal (i.p.) infection withEscherichia coliin lipopolysaccharide (LPS)-responsive C3H/HeN mice but not in LPS-hyporesponsive C3H/HeJ mice. The γδ T cells preferentially expressed invariant Vγ6 and Vδ1 chains and proliferated to produce a large amount of gamma interferon in the presence of LPS. Mice depleted of γδ T cells by T-cell receptor δ gene mutation showed impaired resistance againstE. colias assessed by bacterial growth. Macrophages from C3H/HeN mice infected withE. coliexpressed higher levels of interleukin-15 (IL-15) mRNA than those from the infected C3H/HeJ mice. Administration of anti-IL-15 monoclonal antibody inhibited, albeit partially, the appearance of γδ T cells in C3H/HeN mice afterE. coliinfection and diminished the host defense against the infection. These results suggest that LPS-stimulated γδ T cells play an important role in the host defense againstE. coliinfection and that IL-15 may be partly involved in the protection via an increase in the γδ T cells.
Nonspecific Early Protective Immunity inFrancisellaandListeriaInfections Can Be Dependent on Lymphocytes
Authors: Elkins Karen L. , MacIntyre Ann T. , Rhinehart-Jones Tonya R.
Published: December 2019

Normal mice, but not lymphocyte-deficient or B-cell-deficient mice, given a sublethal infection ofFrancisella tularensisLVS survive a secondary lethal challenge of more than 10,000 50% lethal doses given 3 days later. In this work, we show that similar early protection that is also strongly lymphocyte dependent operates inListeria monocytogenesinfection. Since sublethal infection with either LVS orL. monocytogenesprotects against heterologous lethal challenge, this early protection is nonspecific.
Organization of
Escherichia coli
O157 O Antigen Gene Cluster and Identification of Its Specific Genes
Authors: Wang Lei , Reeves Peter R.
Published: December 2019

The O157:H7 clone of
Escherichia coli
, which causes major, often prolonged outbreaks of gastroenteritis with hemolytic-uremic syndrome (HUS) such as those in Japan, Scotland, and the United States recently, is thought to be resident normally in cattle or other domestic animals. This clone is of major significance for public health and the food industry. We have developed a fast method for sequencing a given O antigen gene cluster and applied it to O157. The O157 O antigen gene cluster is 14 kb in length, comprising 12 genes and a remnant H-repeat unit. Based on sequence similarity, we have identified all the necessary O antigen genes, including five sugar biosynthetic pathway genes, four transferase genes, the O unit flippase gene, and the O antigen polymerase gene. By PCR testing against all 166
E. coli
O serogroups and a range of gram-negative bacterial strains, including some that cross-react serologically with
E. coli
O157 antisera, we have found that certain O antigen genes are highly specific to O157
E. coli
. This work provides the basis for a sensitive test for rapid detection of O157
E. coli
. This is important both for decisions on patient care, since early treatment may reduce the risk of life-threatening complications, and for detection of sources of contamination. The method for fast sequencing of O antigen gene clusters plus an ability to predict which genes will be O antigen specific will enable PCR tests to be developed as needed for other clones of
E. coli
or, once flanking genes are identified, clones of any gram-negative bacterium.
Pulmonary and Hepatic Gene Expression following Cecal Ligation and Puncture: Monophosphoryl Lipid A Prophylaxis Attenuates Sepsis-Induced Cytokine and Chemokine Expression and Neutrophil Infiltration
Authors: Salkowski Cindy A. , Detore Gregory , Franks Alice , Falk Michael C. , Vogel Stefanie N.
Published: December 2019

Polymicrobial sepsis induced by cecal ligation and puncture (CLP) reproduces many of the pathophysiologic features of septic shock. In this study, we demonstrate that mRNA for a broad range of pro- and anti-inflammatory cytokine and chemokine genes are temporally regulated after CLP in the lung and liver. We also assessed whether prophylactic administration of monophosphoryl lipid A (MPL), a nontoxic derivative of lipopolysaccharide (LPS) that induces endotoxin tolerance and attenuates the sepsis syndrome in mice after CLP, would alter tissue-specific gene expression post-CLP. Levels of pulmonary interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), granulocyte colony-stimulating factor (G-CSF), IL-1 receptor antagonist (IL-1ra), and IL-10 mRNA, as well as hepatic IL-1β, IL-6, gamma interferon (IFN-γ), G-CSF, inducible nitric oxide synthase, and IL-10 mRNA, were reduced in MPL-pretreated mice after CLP compared to control mice. Chemokine mRNA expression was also profoundly mitigated in MPL-pretreated mice after CLP. Specifically, levels of pulmonary and hepatic macrophage inflammatory protein 1α (MIP-1α), MIP-1β, MIP-2, and monocyte chemoattractant protein-1 (MCP-1) mRNA, as well as hepatic IFN-γ-inducible protein 10 and KC mRNA, were attenuated in MPL-pretreated mice after CLP. Attenuated levels of IL-6, TNF-α, MCP-1, MIP-1α, and MIP-2 in serum also were observed in MPL-pretreated mice after CLP. Diminished pulmonary chemokine mRNA production was associated with reduced neutrophil margination and pulmonary myeloperoxidase activity. These data suggest that prophylactic administration of MPL mitigates the sepsis syndrome by reducing chemokine production and the recruitment of inflammatory cells into tissues, thereby attenuating the production of proinflammatory cytokines.
Use ofMycobacterium tuberculosisComplex-Specific Antigen Cocktails for a Skin Test Specific for Tuberculosis
Authors: Lyashchenko Konstantin , Manca Claudia , Colangeli Roberto , Heijbel Anna , Williams Alan , Gennaro Maria Laura
Published: December 2019

The tuberculin skin test currently used to diagnose infection withMycobacterium tuberculosishas poor diagnostic value, especially in geographic areas where the prevalence of tuberculosis is low or where the environmental burden of saprophytic, nontuberculous mycobacteria is high. Inaccuracy of the tuberculin skin test often reflects a low diagnostic specificity due to the presence in tuberculin of antigens shared by many mycobacterial species. Thus, a skin test specific for tuberculosis requires the development of new tuberculins consisting of antigens specific toM. tuberculosis. We have formulated cocktails of two to eight antigens ofM. tuberculosispurified from recombinantEscherichia coli. Multiantigen cocktails were evaluated by skin testing guinea pigs sensitized withM. bovisBCG. Reactivity of multiantigen cocktails was greater than that of any single antigen. Cocktail activity increased with the number of antigens in the cocktail even when the same amount of total protein was used for cocktails and for each single antigen. A cocktail of four purified antigens specific for theM. tuberculosiscomplex elicited skin test responses only in BCG-immunized guinea pigs, not in control animals immunized withM. avium. These findings open the way to designing a multiantigen formulation for a skin test specific for tuberculosis.
Synthesis of Microcapsule byStaphylococcus aureusIs Not Responsive to Environmental Phosphate Concentrations
Authors: Fox Karen F. , Stewart George C. , Fox Alvin
Published: December 2019

The polysaccharide microcapsule ofStaphylococcus aureushas been reported to be differentially expressed depending on growth conditions, with phosphate concentration being the critical environmental component. This study evaluated the effect of growth of a serotype 8 strain ofS. aureusin phosphate-replete and phosphate-limiting media on microcapsule production. The presence of the cell wall polymers microcapsule and teichoic acid was measured by both gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry. Production of microcapsule was unaffected by changes in the environmental phosphate concentration. There was, additionally, no evidence for a shift from teichoic acid to teichuronic acid synthesis.
Type-Specific Contributions to Chromosome Size Differences in
Escherichia coli
Authors: Rode Christopher K. , Melkerson-Watson Lyla J. , Johnson Amanda T. , Bloch Craig A.
Published: December 2019

The
Escherichia coli
genome varies in size from 4.5 to 5.5 Mb. It is unclear whether this variation may be distributed finely throughout the genome or is concentrated at just a few chromosomal loci or on plasmids. Further, the functional correlates of size variation in different genome copies are largely unexplored. We carried out comparative macrorestriction mapping using rare-restriction-site alleles (made with the Tn
10
dRCP2 family of elements, containing the
Not
I,
Bln
I, I-
Ceu
I, and ultra-rare-cutting I-
Sce
I sites) among the chromosomes of laboratory
E. coli
K-12, newborn-sepsis-associated
E. coli
RS218, and uropathogenic
E. coli
J96. These comparisons showed just a few large accessory chromosomal segments accounting for nearly all strain-to-strain size differences. Of 10 sepsis-associated and urovirulence genes, previously isolated from the two pathogens by scoring for function, all were colocalized exclusively with one or more of the accessory chromosomal segments. The accessory chromosomal segments detected in the pathogenic strains from physical, macrorestriction comparisons may be a source of new virulence genes, not yet isolated by function.
Influence of Outer Surface Protein A Antibody onBorrelia burgdorferiwithin Feeding Ticks
Authors: de Silva Aravinda M. , Zeidner Nordin S. , Zhang Yan , Dolan Marc C. , Piesman Joseph , Fikrig Erol
Published: December 2019

Borrelia burgdorferi, the spirochetal agent of Lyme disease, is transmitted byIxodesticks. When an infected nymphal tick feeds on a host, the bacteria increase in number within the tick, after which they invade the tick’s salivary glands and infect the host. Antibodies directed against outer surface protein A (OspA) ofB. burgdorferikill spirochetes within feeding ticks and block transmission to the host. In the studies presented here, passive antibody transfer experiments were carried out to determine the OspA antibody titer required to block transmission to the rodent host. OspA antibody levels were determined by using a competitive enzyme-linked immunosorbent assay that measured antibody binding to a protective epitope defined by monoclonal antibody C3.78. The C3.78 OspA antibody titer (>213 μg/ml) required to eradicate spirochetes from feeding ticks was considerably higher than the titer (>6 μg/ml) required to block transmission to the host. Although spirochetes were not eradicated from ticks at lower antibody levels, the antibodies reduced the number of spirochetes within the feeding ticks and interfered with the ability of spirochetes to induceospCand invade the salivary glands of the vector. OspA antibodies may directly interfere with the ability ofB. burgdorferito invade the salivary glands of the vector; alternately, OspA antibodies may lower the density of spirochetes within feeding ticks below a critical threshold required for initiating events linked to transmission.
Five Homologous Repeats of the Protein G-Related Protein MIG Cooperate in Binding to Goat Immunoglobulin G
Authors: Vasi József , Svensson Jan , Frick Inga-Maria , Müller Hans-Peter
Published: December 2019

Protein MIG, fromStreptococcus dysgalactiae, binds α2-macroglobulin and immunoglobulin G (IgG). MIG-derived fusion proteins with one to five IgG-binding repeats differed up to 72,000-fold in avidity for goat IgG, indicating a considerable cooperativity of the repeats. Significant sequence variation in the IgG-binding repeats was recognized. Protein MIG interacted with goat IgG1 via both the Fc and Fab parts.