bluuebunny/crossref_metadata_2025 huggingface dataset:
The
Salmonella enterica
Serotype Typhimurium
lpf
,
bcf
,
stb
,
stc
,
std
, and
sth
Fimbrial Operons Are Required for Intestinal Persistence in Mice
Authors: Weening Eric H. , Barker Jared D. , Laarakker Marijke C. , Humphries Andrea D. , Tsolis Renée M. , Bäumler Andreas J.
Published: May 2005

Salmonella enterica
serotype Typhimurium causes human infections that can frequently be traced back through the food chain to healthy livestock whose intestine is colonized by the pathogen. Little is known about the genes important for intestinal carriage of
S. enterica
serotype Typhimurium in vertebrate animals. Here we characterized the role of 10 fimbrial operons,
agf
,
fim
,
lpf
,
pef
,
bcf
,
stb
,
stc
,
std
,
stf
, and
sth
, using competitive infection experiments performed in genetically susceptible (BALB/c) and resistant (CBA) mice. Deletion of
agfAB
,
fimAICDHF
,
lpfABCDE
,
pefABCDI
,
bcfABCDEFG
,
stbABCD
,
stcABCD
,
stdAB
,
stfACDEFG
, or
sthABCDE
did not reduce the ability of
S. enterica
serotype Typhimurium to colonize the spleen and cecum of BALB/c mice 5 days after infection. Similarly, deletion of
agfAB
,
fimAICDHF
,
pefABCDI
, and
stfACDEFG
did not result in reduced recovery of
S. enterica
serotype Typhimurium from fecal samples collected from infected CBA mice over a 30-day time period. However,
S. enterica
serotype Typhimurium strains carrying deletions in
lpfABCDE
,
bcfABCDEFG
,
stbABCD
,
stcABCD
,
stdAB
, or
sthABCDE
were recovered at significantly reduced numbers from the feces of CBA mice. There was a good correlation (
R
2
= 0.9626) between competitive indices in the cecum and fecal samples of CBA mice at 30 days after infection, suggesting that the recovery of
S. enterica
serotype Typhimurium from fecal samples closely reflected its ability to colonize the cecum. Collectively, these data show that six fimbrial operons (
lpf
,
bcf
,
stb
,
stc
,
std
, and
sth
) contribute to long-term intestinal carriage of
S. enterica
serotype Typhimurium in genetically resistant mice.
The Vi Capsular Antigen ofSalmonella entericaSerotype Typhi Reduces Toll-Like Receptor-Dependent Interleukin-8 Expression in the Intestinal Mucosa
Authors: Raffatellu Manuela , Chessa Daniela , Wilson R. Paul , Dusold Richard , Rubino Salvatore , Bäumler Andreas J.
Published: May 2005

Human infections with nontyphoidalSalmonellaserotypes, such asS. entericaserotype Typhimurium, are characterized by a massive neutrophil influx in the colon and terminal ileum. In contrast, neutrophils are scarce in intestinal infiltrates of typhoid fever patients. Here, we show that inS. entericaserotype Typhi, the causative agent of typhoid fever, expression of the Vi capsular antigen reduced expression of the neutrophil chemoattractant interleukin-8 (IL-8) in host cells. Capsulated bacteria elicited IL-8 expression in polarized human epithelial cells (T84) and human macrophage-like cells (THP-1) in vitro at significantly reduced levels compared to noncapsulated bacteria. Experiments with a human cell line (HEK293) transfected with human Toll-like receptors (TLRs) demonstrated that in the presence of TLR5 or TLR4/MD2/CD14, a noncapsulated serotype Typhi mutant was able to induce the expression of IL-8, while this host response was significantly reduced when cells were infected with the capsulated serotype Typhi wild type. The relevance of these in vitro observations for the interaction of serotype Typhi with its human host was further studied ex vivo using human colonic tissue explants. Expression of IL-8 was detected in human colonic tissue explants infected with serotype Typhimurium or a noncapsulated serotype Typhi mutant. In contrast, infection with the serotype Typhi wild type did not elicit IL-8 expression in colonic tissue explants. Collectively, these data suggest that the scarcity of neutrophils in intestinal infiltrates of typhoid fever patients is due to a capsule-mediated reduction of TLR-dependent IL-8 production in the intestinal mucosa.
Increased Sensitivity to Staphylococcal Enterotoxin B following Adenoviral Infection
Authors: Yarovinsky Timur O. , Mohning Michael P. , Bradford Mary A. , Monick Martha M. , Hunninghake Gary W.
Published: May 2005

Staphylococcal enterotoxin B induces toxic shock and is a major virulence factor of staphylococcal diseases. We examined the effects of systemic adenoviral infection on responses to staphylococcal enterotoxin B in a murine model. We found that adenoviral infection markedly increases the severity of liver injury following exposure to staphylococcal enterotoxin B withoutd-galactosamine sensitization. In adenovirus-infected mice, staphylococcal enterotoxin B triggered a more profound hypothermia and increased apoptosis in the liver. Consistent with these observations, we also found that adenoviral infection primed for an increased production of gamma interferon in vivo and in vitro following stimulation with staphylococcal enterotoxin B. Gamma-interferon-knockout mice did not show increased sensitivity to staphylococcal enterotoxin B following adenoviral infection. These data suggest that a preexisting viral infection primes mice for subsequent staphylococcal enterotoxin B exposure, possibly via a gamma-interferon-mediated mechanism.
Diminished Monocyte Function in Microfilaremic Patients with Lymphatic Filariasis and Its Relationship to Altered Lymphoproliferative Responses
Authors: Sasisekhar B. , Aparna M. , Augustin D. J. , Kaliraj P. , Kar S. K. , Nutman T. B. , Narayanan R. B.
Published: May 2005

Antigen-specific hyporesponsiveness to filarial antigens is a phenomenon observed in patent infection with lymph-dwelling filarial parasites of humans. This phenomenon has been attributed to a multitude of factors, one of which is altered monocyte function. To examine the role played by monocytes in filarial infection, we assessed the responses of monocytes obtained from normal and filarial parasite-infected individuals to both crude filarial antigen and purified recombinant filarial antigen WbSXP-1 and attempted to relate these to the altered lymphoproliferative responses seen in filarial infection. Monocytes from microfilaremic (MF) patients demonstrated an inability to respond to lipopolysaccharide compared to monocytes from endemic normal persons or from lymphedema patients. Indeed, interleukin 1β (IL-1β) production was severely limited, a finding that did not extend to monocyte responses to filarial antigens. Serum from MF patients reduced adherence and spreading of normal monocytes, a finding not seen with serum from the other clinical groups. Interestingly, there was a significant correlation between the production of IL-1β and adherence. Moreover, the levels of spontaneous production of IL-1β correlated with high levels of spontaneous secretion of IL-10. The effects observed were not a result of diminished viability or alteration in the expression of the cell surface markers CD14 and HLA-DR. These data suggest that monocyte function is dampened in MF patients, a finding which could alter lymphoproliferative responses during patent infection.
Transcriptional Control of Impaired Th1 Responses in Patent Lymphatic Filariasis by T-Box Expressed in T Cells and Suppressor of Cytokine Signaling Genes
Authors: Babu Subash , Kumaraswami V. , Nutman Thomas B.
Published: May 2005

T-bet (T-box expressed in T cells) and GATA-3 are transcription factors that play a critical role in the development of Th1 and Th2 cells, as do genes of the SOCS (suppressor of cytokine signaling) family, albeit indirectly. Another transcription factor, Foxp3, is a master regulator of natural regulatory T cells (Tregs). To identify the role of these factors in impaired Th1 responses of patent filarial infection, analysis of cytokine, SOCS, and transcription factor mRNA expression was performed on purified T cells of filaria-infected individuals (n= 6) and uninfected controls (n= 6). As expected (and in contrast to cells of uninfected individuals), there was a significant depression of gamma interferon (IFN-γ) and a concomitant increase in interleukin-4 (IL-4), IL-5, and IL-10 mRNA expression following stimulation with parasite antigen (BmA) but not with a polyclonal T-cell (anti-CD3) stimulus. T-bet (but not GATA-3) was expressed at significantly lower levels in cells of filaria-infected individuals in response to BmA compared with those from the uninfected group, accounting, at least partially, for the diminished IFN-γ expression. Second, we found no significant differences in expression of Foxp3 between the two groups, although induction of Foxp3 expression correlated with induced expression levels of IL-10, implicating Tregs in the IL-10 expression seen. Finally, parasite-specific T-cell expression of SOCS-1, SOCS-5, and SOCS-7 was significantly diminished among infected patients; in contrast, expression of SOCS-3 increased. Our data therefore indicate that the impaired Th1 responses observed in patent lymphatic filariasis are associated with decreased expression of T-bet, SOCS-1, SOCS-5, and SOCS-7 and increased expression of SOCS-3 in T cells.
Host Cytokine Production, Lymphoproliferation, and Antibody Responses during the Course of
Ancylostoma ceylanicum
Infection in the Golden Syrian Hamster
Authors: Mendez Susana , Valenzuela Jesus G. , Wu Wenhui , Hotez Peter J.
Published: May 2005

The Syrian Golden hamster (
Mesocricetus auratus
) has been used to model infections with the hookworm
Ancylostoma ceylanicum
. New molecular immunological reagents to measure cellular immune responses in hamsters were developed and used to determine the impact of
A. ceylanicum
hookworm infection on host cytokine responses and lymphoproliferation. Initial larval infection with 100 third-stage
A. ceylanicum
larvae resulted in predominant Th1 responses (upregulation of proinflammatory cytokines) that lasted for the duration of larval migration and continued up to 14 days postinfection (prepatency). Subsequently, development of larvae into egg-laying adult hookworms (patency) coincided with a switch to Th2 predominant responses (interleukin-4 [IL-4]) as well as a marked increase in IL-10 production. This switch also concurred with reduced host lymphoproliferative responses to hookworm antigens. The findings demonstrate a similarity in immune responses between hamsters and humans infected with hookworms, suggesting that hamsters will be a useful animal model species for examining host immunity to human hookworm infections.
Combining Anthrax Vaccine and Therapy: a Dominant-Negative Inhibitor of Anthrax Toxin Is Also a Potent and Safe Immunogen for Vaccines
Authors: Aulinger Benedikt A. , Roehrl Michael H. , Mekalanos John J. , Collier R. John , Wang Julia Y.
Published: May 2005

Anthrax is caused by the unimpeded growth of
Bacillus
anthracis
in the host and the secretion of toxins. The currently available vaccine is based on protective antigen (PA), a central component of anthrax toxin. Vaccination with PA raises no direct immune response against the bacilli and, being a natural toxin component, PA might be hazardous when used immediately following exposure to
B. anthracis
. Thus, we have sought to develop a vaccine or therapeutic agent that is safe and eliminates both secreted toxins and bacilli. To that end, we have previously developed a dually active vaccine by conjugating the capsular poly-γ-
d
-glutamate (PGA) with PA to elicit the production of antibodies specific for both bacilli and toxins. In the present report, we describe the improved potency of anthrax vaccines through the use of a dominant-negative inhibitory (DNI) mutant to replace PA in PA or PA-PGA vaccines. When tested in mice, DNI alone is more immunogenic than PA, and DNI-PGA conjugate elicits significantly higher levels of antibodies against PA and PGA than PA-PGA conjugate. To explain the enhanced immunogenicity of DNI, we propose that the two point mutations in DNI may have improved epitopes of PA allowing better antigen presentation to helper T cells. Alternatively, these mutations may enhance the immunological processing of PA by altering endosomal trafficking of the toxin in antigen-presenting cells. Because DNI has previously been demonstrated to inhibit anthrax toxin, postexposure use of DNI-based vaccines, including conjugate vaccines, may provide improved immunogenicity and therapeutic activity simultaneously.
Role of
Staphylococcus aureus
Global Regulators
sae
and σ
B
in Virulence Gene Expression during Device-Related Infection
Authors: Goerke Christiane , Fluckiger Ursula , Steinhuber Andrea , Bisanzio Vittoria , Ulrich Martina , Bischoff Markus , Patti Joseph M. , Wolz Christiane
Published: May 2005

The ability of
Staphylococcus aureus
to adapt to different environments is due to a regulatory network comprising several loci. Here we present a detailed study of the interaction between the two global regulators
sae
and σ
B
of
S. aureus
and their influence on virulence gene expression in vitro, as well as during device-related infection. The expression of
sae
,
asp23
,
hla
,
clfA
,
coa
, and
fnbA
was determined in strain Newman and its isogenic
saeS/R
and
sigB
mutants by Northern analysis and LightCycler reverse transcription-PCR. There was no indication of direct cross talk between the two regulators.
sae
had a dominant effect on target gene expression during device-related infection. σ
B
seemed to be less active throughout the infection than under induced conditions in vitro.
Entamoeba histolyticaandEntamoeba disparUtilize Externalized Phosphatidylserine for Recognition and Phagocytosis of Erythrocytes
Authors: Boettner Douglas R. , Huston Christopher D. , Sullivan James A. , Petri William A.
Published: May 2005

Amebic erythrophagocytosis is characteristic of invasive amebiasis, and mutants deficient in erythrocyte ingestion are avirulent. We sought to understand the molecular mechanisms underlying erythrocyte phagocytosis byEntamoeba histolytica. Following adherence to amebae, erythrocytes became round and crenulated, and phosphatidylserine (PS) was exposed on their outer membrane leaflets. These changes were similar to the effects of calcium treatment on erythrocytes, which we utilized to separate ameba-induced exposure of erythrocyte PS from the process of phagocytosis. The adherence and phagocytosis of calcium-treated erythrocytes were less inhibited by galactose than were those of healthy erythrocytes, suggesting the existence of an amebic coreceptor specific for PS. To test whether PS was recognized by amebae, calcium-treated cells were incubated with annexin V prior to adherence to or ingestion byE. histolytica. Annexin V blocked both adherence (50% ± 12% inhibition;P< 0.05) and phagocytosis (65% ± 10%;P< 0.05), providing evidence that at least one galactose-independent coreceptor was involved in the adherence and ingestion of red blood cells. The coreceptor was inhibited by phospho-l-serine and to a lesser extent by phospho-d-serine but not by phospho-l-threonine, which is consistent with the coreceptor functioning in the adherence and ingestion of erythrocytes via recognition of PS. We expanded our investigations to the highly related but noninvasive parasiteEntamoeba disparand demonstrated that it was deficient in red-blood-cell adherence, induction of PS exposure, and phagocytosis. These findings establish phosphatidylserine involvement in erythrophagocytosis by amebae and suggest the existence of a PS receptor on the surfaces of bothE. histolyticaandE. dispar.
Killing ofdsrAMutants ofHaemophilus ducreyiby Normal Human Serum Occurs via the Classical Complement Pathway and Is Initiated by Immunoglobulin M Binding
Authors: Abdullah Malikah , Nepluev Igor , Afonina Galyna , Ram Sanjay , Rice Peter , Cade William , Elkins Christopher
Published: May 2005

Previously, we showed that serum resistance inHaemophilus ducreyitype strain 35000HP required expression of the outer membrane protein DsrA because the isogenicdsrAmutant FX517 is highly serum susceptible. In this study, we confirmed this finding by construction of additional serum-susceptibledsrA mutants in more recently isolated serum-resistant strains. We also demonstrated that killing ofdsrAmutants required an intact classical complement cascade but not the alternative or mannan-binding lectin pathways. Between 5- and 10-fold more purified human immunoglobulin M (IgM) but not IgG was deposited ontodsrAmutant FX517 than onto parent strain 35000HP, consistent with IgM initiation of the classical cascade. Depletion of IgM, but not IgG, from complement-intact serum inhibited killing of FX517. As predicted from the amounts of IgM bound, more of the individual complement components were bound by FX517 than by parent strain 35000HP. Examination of the binding of negative regulators of complement as an explanation for serum resistance indicated that parent strain 35000HP bound more C4 binding protein and vitronectin than FX517 but not factor H. However, the degree and pattern of complement component binding observed suggested that IgM binding to the serum-susceptible mutant FX517 was responsible for the activation of the classical pathway and the observed killing of FX517 as opposed to binding of negative regulators of complement by the serum-resistant parent. We speculate that an undefined neo-epitope, possibly carbohydrate, is exposed in thedsrAmutant that is recognized by naturally occurring bactericidal IgM antibodies present in human sera.
Coinfection withAnaplasma phagocytophilumAltersBorrelia burgdorferiPopulation Distribution in C3H/HeN Mice
Authors: Holden Kevin , Hodzic Emir , Feng Sunlian , Freet Kimberly J. , Lefebvre Rance B. , Barthold Stephen W.
Published: May 2005

Borrelia burgdorferi, the agent of Lyme disease, andAnaplasma phagocytophilum, the agent of human anaplasmosis, are both transmitted byIxodessp. ticks and may occasionally coinfect a host. The population distributions of tick-transmittedB. burgdorferiinfection were assessed using quantitative PCR targeting theflaBgene ofB. burgdorferiin the ear, heart base, quadriceps muscle, skin, and tibiotarsal joint tissue of C3H mice previously infected withA. phagocytophilum. Population distributions ofAnaplasmainfection were assessed by targeting thep44gene.A. phagocytophilumin blood and serologic response to both agents were evaluated. Spirochete numbers were increased in the ears, heart base, and skin of coinfected mice, butAnaplasmanumbers remained constant. Antibody response toA. phagocytophilum, but notB. burgdorferi, was decreased in coinfected mice. These results suggest that coinfection withA. phagocytophilumandB. burgdorferimodulates pathogen burden and host antibody responses. This may be explained by the ability ofA. phagocytophilumto functionally impair neutrophils, important cells in the early defense againstB. burgdorferiinfection.
Gamma Interferon Enhances Internalization and Early Nonoxidative Killing ofSalmonella entericaSerovar Typhimurium by Human Macrophages and Modifies Cytokine Responses
Authors: Gordon Melita A. , Jack Dominic L. , Dockrell David H. , Lee Margaret E. , Read Robert C.
Published: May 2005

Gamma interferon (IFN-γ) is a critical cytokine in host defense against salmonella infections, but its role in phagocytic killing of intracellularSalmonellaspp. has been investigated mainly in animal rather than human cells. We measured the effect of recombinant IFN-γ (rIFN-γ) priming on bacterial internalization, intracellular killing, oxidative burst, and cytokine release during phagocytosis ofSalmonella entericaserovar Typhimurium by human monocyte-derived macrophages (MDM). Eleven-day-old MDM, primed for 72 h with rIFN-γ (100 ng/ml) exhibited an increased proportion of cells with associated bacteria (31% versus 26%,P= 0.036) and a 67% increase in internalized bacteria per cell compared to unprimed cells (P= 0.025). Retrieval of viable bacteria following internalization was reduced 3.6-fold in 72-h primed versus unprimed MDM (interquartile range, 3.1 to 6.4) at 0.5 h due to enhanced early intracellular killing, and this difference was maintained up to 24 h. In contrast, cells primed for only 24 h exhibited no increase in early killing. MDM were competent to produce an early oxidative burst when stimulated with phorbol myristate acetate, which was fully abrogated by the respiratory burst inhibitor diphenyleneiodonium chloride (DPI), but infection of MDM withS. entericaserovar Typhimurium did not cause an increase in the early respiratory burst under unprimed or primed conditions, and DPI had no effect on the early killing of bacteria by primed or unprimed MDM. During 24 h following infection, rIFN-γ-primed MDM released more interleukin-12 (IL-12) and less IL-10 relative to unprimed cells. We conclude that 72-h priming with rIFN-γ increases the efficiency of internalization and nonoxidative early intracellular killing ofS. entericaserovar Typhimurium by human macrophages and modifies subsequent cytokine release.
Gamma Interferon Positively ModulatesActinobacillus actinomycetemcomitans-Specific RANKL+CD4+Th-Cell-Mediated Alveolar Bone Destruction In Vivo
Authors: Teng Yen-Tung A. , Mahamed Deeqa , Singh Bhagirath
Published: May 2005

Recent studies have shown the biological and clinical significance of signaling pathways of osteogenic cytokines RANKL-RANK/OPG in controlling osteoclastogenesis associated with bone pathologies, including rheumatoid arthritis, osteoporosis, and other osteolytic disorders. In contrast to the inhibitory effect of gamma interferon (IFN-γ) on RANKL-mediated osteoclastogenesis reported recently, alternative new evidence is demonstrated via studies of experimental periodontitis using humanized NOD/SCID and diabetic NOD mice and clinical human T-cell isolates from diseased periodontal tissues, where the presence of increasing IFN-γ is clearly associated with (i) enhancedActinobacillus actinomycetemcomitans-specific RANKL-expressing CD4+Th cell-mediated alveolar bone loss during the progression of periodontal disease and (ii) a concomitant and significantly increased coexpression of IFN-γ in RANKL(+) CD4+Th cells. Therefore, there are more complex networks in regulating RANKL-RANK/OPG signaling pathways for osteoclastogenesis in vivo than have been suggested to date.
Distinct Th1- and Th2-Type Prenatal Cytokine Responses toPlasmodium falciparumErythrocyte Invasion Ligands
Authors: Malhotra Indu , Mungai Peter , Muchiri Eric , Ouma John , Sharma Shobhona , Kazura James W. , King Christopher L.
Published: May 2005

Prenatal immunity toPlasmodium falciparummerozoite proteins involved in erythrocyte invasion may contribute to the partial protection against malaria that is acquired during infancy in areas of stable malaria transmission. We examined newborn and maternal cytokine and antibody responses to merozoite surface protein-1 (MSP-1), ribosomal phosphoprotein P0 (PfP0), and region II of erythrocyte binding antigen-175 (EBA-175) in infant-mother pairs in Kenya. Overall, 82 of 167 (50%), 106 of 176 (60%), and 38 of 84 (45%) cord blood lymphocytes (CBL) from newborns produced one or more cytokines in response to MSP-1, PfP0, and EBA-175, respectively. Newborns of primigravid and/or malaria-infected women were more likely to have antigen-responsive CBL than were newborns of multigravid and/or uninfected women at delivery. Newborn cytokine responses did not match those of their mothers and fell into three distinct categories, Th1 (21 of 55 CBL donors produced only gamma interferon and/or interleukin 2 [IL-2]), Th2 (21 of 55 produced only IL-5 and/or IL-13), and mixed Th1/Th2 (13 of 55). Newborns produced more IL-10 than adults. High and low levels of cord blood IL-12 p70 production induced by anti-CD40 activation were associated with malaria-specific Th1 and Th2 responses, respectively. Antigen-responsive CBL in some newborns were detected only after depletion of IL-10-secreting CD8 cells with enrichment for CD4 cells. These data indicate that prenatal sensitization to blood-stagePlasmodium falciparumoccurs frequently in areas where malaria is holoendemic. Modulation of this immunity, possibly by maternal parity and malaria, may affect the acquisition of protective immunity against malaria during infancy.
Identification of Surface Antigens of
Moraxella catarrhalis
as Targets of Human Serum Antibody Responses in Chronic Obstructive Pulmonary Disease
Authors: Murphy Timothy F. , Brauer Aimee L. , Aebi Christoph , Sethi Sanjay
Published: May 2005

Moraxella catarrhalis
is an important respiratory tract pathogen, causing otitis media in children and lower respiratory tract infections in adults with chronic obstructive pulmonary disease (COPD). Adults with COPD make antibody responses to
M. catarrhalis
following infection, but little is known about the identity of the antigens to which these antibodies are directed. In this study, 12 serum samples obtained from adults with COPD who had cleared
M. catarrhalis
from the respiratory tract following infection and who had developed new serum immunoglobulin G (IgG) to their infecting strain were subjected to a series of assays to identify the antigens to which potentially protective antibodies were directed. Sera were adsorbed with intact bacterial cells, and antibodies were eluted from the surfaces of the bacteria. Analysis by flow cytometry established that adsorption and elution effectively detected antibodies specifically directed to surface-exposed epitopes. Immunoblot assays of adsorbed and eluted serum fractions were performed with purified outer membranes and purified lipooligosaccharide of homologous infecting strains and with a series of mutants deficient in expression of individual outer membrane proteins (OMPs). While heterogeneity in antibody responses among individuals was observed, five major OMPs, UspA1, UspA2, Hag, TbpB, and OMP CD, were identified as targets of antibodies to surface epitopes in the majority of adults with COPD who cleared the organism. These results have important implications in understanding human immune responses to
M. catarrhalis
and in elucidating the elements of a protective immune response.
Identification, Distribution, and Expression of Novel Genes in 10 Clinical Isolates of Nontypeable
Haemophilus influenzae
Authors: Shen Kai , Antalis Patricia , Gladitz John , Sayeed Sameera , Ahmed Azad , Yu Shujun , Hayes Jay , Johnson Sandra , Dice Bethany , Dopico Richard , Keefe Randy , Janto Benjamin , Chong William , Goodwin Joseph , Wadowsky Robert M. , Erdos Geza , Post J. Christopher , Ehrlich Garth D. , Hu Fen Z.
Published: May 2005

We hypothesize that
Haemophilus influenzae
, as a species, possesses a much greater number of genes than that found in any single
H. influenzae
genome. This supragenome is distributed throughout naturally occurring infectious populations, and new strains arise through autocompetence and autotransformation systems. The effect is that
H. influenzae
populations can readily adapt to environmental stressors. The supragenome hypothesis predicts that significant differences exist between and among the genomes of individual infectious strains of nontypeable
H.
influenzae
(NTHi). To test this prediction, we obtained 10 low-passage NTHi clinical isolates from the middle ear effusions of patients with chronic otitis media. DNA sequencing was performed with 771 clones chosen at random from a pooled genomic library. Homology searching demonstrated that ∼10% of these clones were novel compared to the
H. influenzae
Rd KW20 genome, and most of them did not match any DNA sequence in GenBank. Amino acid homology searches using hypothetical translations of the open reading frames revealed homologies to a variety of proteins, including bacterial virulence factors not previously identified in the NTHi isolates. The distribution and expression of 53 of these genes among the 10 strains were determined by PCR- and reverse transcription PCR-based analyses. These unique genes were nonuniformly distributed among the 10 isolates, and transcription of these genes in planktonic cultures was detected in 50% (177 of 352) of the occurrences. All of the novel sequences were transcribed in one or more of the NTHi isolates. Seventeen percent (9 of 53) of the novel genes were identified in all 10 NTHi strains, with each of the remaining 44 being present in only a subset of the strains. These genic distribution analyses were more effective as a strain discrimination tool than either multilocus sequence typing or 23S ribosomal gene typing methods.
Contribution of theMycobacterium tuberculosisMmpL Protein Family to Virulence and Drug Resistance
Authors: Domenech Pilar , Reed Michael B. , Barry Clifton E.
Published: May 2005

The genome sequence ofMycobacterium tuberculosisrevealed the presence of 12 membrane proteins proposed to have a function in the transport of lipids. Insertional inactivation of 11 of these has revealed that only 1 (MmpL3) is apparently essential for viability. Five of these proteins are conserved within the genome ofMycobacterium leprae. The drug susceptibilities of these 11 mutants to a broad spectrum of agents are unaltered, suggesting that unlike their function in other organisms, these proteins do not play a significant role in intrinsic drug resistance. Each of these mutants was assessed for growth kinetics and lethality in a murine low-dose aerosol model of tuberculosis, and four were found to be impaired in one or both measures of virulence. Two of these, with mutations of MmpL4 and the previously characterized MmpL7, which transports phthiocerol dimycocerosate, were found to have both impaired growth kinetics and impaired lethality. Mutants with inactivation of MmpL8, which transports a precursor of the sulfatides, or MmpL11, which transports an unknown substrate, were found to establish infection normally but to be significantly attenuated for lethality in time-to-death studies. These studies support the concept that MmpL-mediated lipid secretion both contributes to the innate ability of the pathogen to survive intracellularly and also contributes directly to the host-pathogen dialogue that determines the ultimate outcome of infection.
Staphylococcus aureusStrains That Express Serotype 5 or Serotype 8 Capsular Polysaccharides Differ in Virulence
Authors: Watts Andrew , Ke Danbing , Wang Qun , Pillay Anil , Nicholson-Weller Anne , Lee Jean C.
Published: May 2005

Most isolates ofStaphylococcus aureusproduce a serotype 5 (CP5) or 8 (CP8) capsular polysaccharide. To investigate whether CP5 and CP8 differ in their biological properties, we created isogenic mutants ofS. aureusReynolds that expressed CP5, CP8, or no capsule. Biochemical analyses of CP5 and CP8 purified from the isogenicS. aureusstrains were consistent with published structures. The degree of O acetylation of each polysaccharide was similar, but CP5 showed a greater degree of N acetylation. Mice challenged with the CP5+strain showed a significantly higher bacteremia level than mice challenged with the CP8+strain. Similarly, the CP5+strain survived preferentially in the bloodstream and kidneys of infected mice challenged with a mixed inoculum containing both strains. The enhanced virulence of the CP5+strain in vivo correlated with its greater resistance to in vitro killing in whole mouse blood. Likewise, in vitro opsonophagocytic killing assays with human neutrophils and sera revealed greater survival of the Reynolds (CP5) strain, even though the kinetics of opsonization by C3b and iC3b was similar for both the CP5+and CP8+strains. Electron micrographs demonstrated C3 molecules on the cell wall beneath the capsule layer for both serotype 5 and 8 strains. Purified CP5 and CP8 stimulated a modest oxidative burst in human neutrophils but failed to activate the alternative complement pathway. These results indicate that CP5 and CP8 differ in a number of biological properties, and these differences likely contribute to the relative virulence of serotype 5 and 8S. aureusin vivo.
Identification and Characterization of an Autolysin-Encoding Gene of
Streptococcus mutans
Authors: Shibata Yukie , Kawada Miki , Nakano Yoshio , Toyoshima Kuniaki , Yamashita Yoshihisa
Published: May 2005

We identified a gene (
atlA
) encoding autolytic activity from
Streptococcus mutans
Xc. The AtlA protein predicted to be encoded by
atlA
is composed of 979 amino acids with a molecular weight of 107,279 and has a conserved β-1,4-
N
-acetylmuramidase (lysozyme) domain in the C-terminal portion. Sodium dodecyl sulfate extracts of strain Xc showed two major bacteriolytic bands with molecular masses of 107 and 79 kDa, both of which were absent from a mutant with inactivated
atlA
. Western blot analysis revealed that the 79-kDa band was derived from the 107-kDa peptide by cleavage of its N-terminal portion. The inactivation of
atlA
resulted in a marked decrease in autolysis and the formation of very long chains of cells compared to the case for the parent strain. Although both the parent and mutant strains formed biofilms in the presence of sucrose, the biofilms formed by the mutant had a sponge-like architecture with large gaps and contained 30% less biomass than those formed by the parent strain. Furthermore, strain Xc formed glucose-dependent, loose biofilms in the absence of sucrose, but the mutant lost this ability. These results suggest that AtlA may play an important role in biofilm formation by
S. mutans
. The antibody produced against the C-terminal peptide containing the β-1,4-
N
-acetylmuramidase domain drastically inhibited the autolytic activity of strain Xc. This inhibition was specific among the oral streptococci to
S. mutans
. These results indicate that the catalytic domain of AtlA is located at the C terminus, suggesting that further characterization of this domain may provide a means to control cariogenic dental plaque formation.
Immunization of Volunteers withSalmonella entericaSerovar Typhi Strain Ty21a Elicits the Oligoclonal Expansion of CD8+T Cells with Predominant Vβ Repertoires
Authors: Salerno-Gonçalves Rosângela , Wahid Rezwanul , Sztein Marcelo B.
Published: May 2005

CD8+T cells are likely to play an important role in host defense againstSalmonella entericaserovar Typhi by several effector mechanisms, including lysis of infected cells (cytotoxicity) and gamma interferon (IFN-γ) secretion. In an effort to better understand these responses, we studied the T-cell receptor (TCR) repertoire of serovar Typhi-specific CD8+T cells in humans. To this end, we determined the TCR beta chain (Vβ) usage of CD8+T cells from three volunteers orally immunized with Ty21a typhoid vaccine by flow cytometry using a panel of monoclonal antibodies. Although TCR Vβ usage varied among volunteers, we identified oligoclonal Vβ subset expansions in individual volunteers (Vβ 2, 5.1, 8, 17, and 22 in volunteer 1; Vβ 1, 2, 5.1, 14, 17, and 22 in volunteer 2; and Vβ 3, 8, 14, and 16 in volunteer 3). These subsets were antigen specific, as shown by cytotoxicity and IFN-γ secretion assays on Vβ sorted cells and on T-cell clones derived from these volunteers. Moreover, eight-color flow cytometric analysis showed that these clones exhibited a T effector memory phenotype (i.e., CCR7−CD27−CD45RO+CD62L−) and coexpressed gut homing molecules (e.g., high levels of integrin α4β7, intermediate levels of CCR9, and low levels of CD103). In conclusion, our results show that long-term T-cell responses to serovar Typhi in Ty21a vaccinees are oligoclonal, involving multiple TCR Vβ families. Moreover, these serovar Typhi-specific CD8+T cells bearing defined Vβ specificities are phenotypically and functionally consistent with T effector memory cells with preferential gut homing potential.
Impairment of Protective Immunity to Blood-Stage Malaria by Concurrent Nematode Infection
Authors: Su Zhong , Segura Mariela , Morgan Kenneth , Loredo-Osti J. Concepcion , Stevenson Mary M.
Published: May 2005

Helminthiases, which are highly prevalent in areas where malaria is endemic, have been shown to modulate or suppress the immune response to unrelated antigens or pathogens. In this study, we established a murine model of coinfection with a gastrointestinal nematode parasite,Heligmosomoides polygyrus, and the blood-stage malaria parasitePlasmodium chabaudiAS in order to investigate the modulation of antimalarial immunity by concurrent nematode infection. Chronic infection with the nematode for 2, 3, or 5 weeks beforeP. chabaudiAS infection severely impaired the ability of C57BL/6 mice to control malaria, as demonstrated by severe mortality and significantly increased malaria peak parasitemia levels. Coinfected mice produced significantly lower levels of gamma interferon (IFN-γ) duringP. chabaudiAS infection than mice infected with malaria alone. Concurrent nematode infection also suppressed production of type 1-associated, malaria-specific immunoglobulin G2a. Mice either infected with the nematode alone or coinfected with the nematode and malaria had high transforming growth factor β1 (TGF-β1) levels, and concurrent nematode and malaria infections resulted in high levels of interleukin-10 in vivo. Splenic CD11c+dendritic cells (DC) from mice infected with malaria alone and coinfected mice showed similarly increased expression of CD40, CD80, and CD86, but DC from coinfected mice were unable to induce CD4+T-cell proliferation and optimal IFN-γ production in response to the malaria antigen in vitro. Importantly, treatment of nematode-infected mice with an anthelmintic drug prior to malaria infection fully restored protective antimalarial immunity and reduced TGF-β1 levels. These results demonstrate that concurrent nematode infection strongly modulates multiple aspects of immunity to blood-stage malaria and consequently impairs the development of protective antimalarial immunity.
Vaccination of Cattle with a CpG Oligodeoxynucleotide-Formulated Mycobacterial Protein Vaccine andMycobacterium bovisBCG Induces Levels of Protection against Bovine Tuberculosis Superior to Those Induced by Vaccination with BCG Alone
Authors: Wedlock D. Neil , Denis Michel , Skinner Margot A. , Koach Jessica , de Lisle Geoffrey W. , Vordermeier H. Martin , Hewinson R. Glyn , van Drunen Littel-van den Hurk Sylvia , Babiuk Lorne A. , Hecker Rolf , Buddle Bryce M.
Published: May 2005

The development of a subunit protein vaccine for bovine tuberculosis which could be used either in combination withMycobacterium bovisBCG (to improve the efficacy of that vaccine) or alone would offer significant advantages over currently available strategies. A study was conducted with cattle to determine the protective efficacy of a strategy based on concurrent immunization with anM. bovisculture filtrate (CFP) vaccine and BCG compared to vaccination with either vaccine alone. One group of calves (10 animals per group) was vaccinated subcutaneously with CFP formulated with Emulsigen and combined with a CpG oligodeoxynucleotide (ODN). A second group was vaccinated with both the CFP vaccine and BCG injected at adjacent sites (CFP-BCG). One further group was vaccinated subcutaneously with BCG, while another group served as nonvaccinated control animals. Vaccination with CFP-BCG induced levels of antigen-specific gamma interferon (IFN-γ) and interleukin-2 (IL-2) in whole-blood cultures that were higher than those induced by vaccination with BCG alone. The combination of CFP and BCG did not enhance the production of antibodies toM. bovisCFP compared to vaccination with CFP alone. Vaccination with CFP alone led to delayed antigen-specific IFN-γ and IL-2 responses. Vaccination with CFP-BCG induced a high level of protection against an intratracheal challenge with virulentM. bovis, based on a significant enhancement of six pathological and microbiological parameters of protection compared with the nonvaccinated group. In contrast, vaccination with BCG alone induced a significant enhancement of protection in only one parameter, while CFP alone induced no protection. These results suggest that a combination of a CpG ODN-formulated protein vaccine and BCG offers better protection against bovine tuberculosis than does BCG alone.
Peripheral Blood and Pleural Fluid Mononuclear Cell Responses to Low-Molecular-Mass Secretory Polypeptides ofMycobacterium tuberculosisin Human Models of Immunity to Tuberculosis
Authors: Sable Suraj B. , Kumar Rajnish , Kalra Mamta , Verma Indu , Khuller G. K. , Dobos Karen , Belisle John T.
Published: May 2005

A total of 104 polypeptides were purified from the low-molecular-mass secretory proteome ofMycobacterium tuberculosisH37Rv using a combination of anion exchange column chromatography and high resolution preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by electroelution. The goal of this study was to identify polypeptides from a low-molecular-mass secretory proteome recognized by human subjects infected withM. tuberculosisand to ascertain the differences in specificity of antigen recognition by the peripheral blood mononuclear cells (PBMCs) and pleural fluid mononuclear cells (PFMCs) of these individuals. The study identified CFP-8 (Rv0496), CFP-11 (Rv2433c), CFP-14.5 (Rv2445c), and CFP-31 (Rv0831c) as novel T-cell antigens apart from previously characterized ESAT-6, TB10.4, CFP10, GroES, MTSP14, MTSP17, CFP21, MPT64, Ag85A, and Ag85B on the basis of recognition by PBMCs of tuberculosis contacts and treated tuberculosis patients. Further, polypeptides prominently recognized by PFMCs of tuberculous pleurisy patients were the same as those recognized by PBMCs of healthy contacts and treated tuberculosis patients. The results of our study indicate the homogeneity of antigenic target recognition by lymphocytes at the site of infection and at the periphery in the human subjects studied and the need to evaluate these antigenic targets as components of future antituberculous vaccines.
Cytolethal Distending Toxin Is Essential forHelicobacter hepaticusColonization in Outbred Swiss Webster Mice
Authors: Ge Zhongming , Feng Yan , Whary Mark T. , Nambiar Prashant R. , Xu Shilu , Ng Vivian , Taylor Nancy S. , Fox James G.
Published: May 2005

Helicobacter hepaticus, which induces chronic hepatitis and typhlocolitis in susceptible mouse strains, produces a cytolethal distending toxin (CDT) consisting of CdtA, CdtB, and CdtC. AcdtB-deficientH. hepaticusisogenic mutant (HhcdtBm7) was generated and characterized for colonization parameters in four intestinal regions (jejunum, ileum, cecum, and colon) of outbred Swiss Webster (SW) mice. Inactivation of thecdtBgene abolished the ability of HhcdtBm7 to colonize female mice at both 8 and 16 weeks postinfection (wpi), whereas HhcdtBm7 colonized all of four intestinal regions of three of five males at 8 wpi and then was eliminated by 16 wpi. Wild-type (WT)H. hepaticuswas detected in the corresponding intestinal regions of both male and female mice at 8 and 16 wpi; however, colonization levels of WTH. hepaticusin the cecum and colon of male mice were approximately 1,000-fold higher than in females (P< 0.0079) at 16 wpi. Infection with WTH. hepaticus, but not HhcdtBm7, at 8 wpi was associated with significantly increased mRNA level of ileal and cecal gamma interferon (IFN-γ) in females (P< 0.016 and 0.031 between WTH. hepaticus-infected and sham-dosed females, respectively). In contrast, the mRNA levels of IFN-γ were significantly higher in the colon (P< 0.0079) and trended to be higher in the cecum (P< 0.15) in the HhcdtBm7-colonized male mice versus the sham-dosed controls at 8 wpi. In addition, mRNA levels of ileal IFN-γ were significantly higher in the control females than males at 8 wpi (P< 0.016). There were significantly higher Th1-associated immunoglobulin G2a (IgG2a), Th2-associated IgG1 and mucosal IgA (P< 0.002, 0.002, 0.002, respectively) responses in the mice infected with WTH. hepaticuswhen compared to HhcdtBm7 at 16 wpi. Colonic interleukin-10 (IL-10) expressions at 16 wpi were significantly lower in both female and male mice colonized by WTH. hepaticusor in males transiently colonized through 8 wpi by HhcdtBm7 versus control mice (P< 0.0159). These lines of evidence indicate that (i)H. hepaticusCDT plays a crucial role in the persistent colonization ofH. hepaticusin SW mice; (ii) SW female mice are more resistant toH. hepaticuscolonization than male mice; (iii) there was persistent colonization of WTH. hepaticusin cecum, colon, and jejunum but only transient colonization ofH. hepaticusin the ileum of female mice; (iv)H. hepaticuscolonization was associated with down-regulation of colonic IL-10 production.
Inducible Antisense RNA Expression in the Characterization of Gene Functions in
Streptococcus mutans
Authors: Wang Bing , Kuramitsu Howard K.
Published: May 2005

In order to examine gene function in
Streptococcus mutans
, we have recently initiated an antisense RNA strategy. Toward this end, we have now constructed and evaluated three
Escherichia coli-S. mutans
shuttle expression vectors with the
fruA
and
scrB
promoters from
S.
mutans
, as well as the
tetR
-controlled
tetO
promoter from
Staphylococcus aureus
. Among these, the
tetO
/
tetR
system proved to be the most tightly controlled promoter. By using this shuttle plasmid system, modulation of gene function by inducible antisense RNA expression was demonstrated for
comC
antisense fragments of different sizes as well as for distinct
gtfB
antisense fragments. It was demonstrated that the size, but not the relative position, of an antisense DNA fragment is important in mediating the antisense phenomenon. Furthermore, by constructing and screening random DNA libraries with the
tet
expression shuttle system, 78 growth-retarded transformants harboring antisense DNA fragments were also identified. Almost all of them corresponded to homologous essential genes in other bacteria. In addition, a novel essential gene, the
coaE
gene, encoding dephospho-coenzyme A kinase, which is involved in the final step of coenzyme A catabolism in
S. mutans
, was identified and characterized. These results suggest that the antisense RNA strategy can be useful for identifying novel essential genes in
S. mutans
bacteria as well as further characterizing the physiology (including potential virulence factors) of these organisms.
Gamma Interferon-Induced T-Cell Loss in VirulentMycobacterium aviumInfection
Authors: Flórido Manuela , Pearl John E. , Solache Alejandra , Borges Margarida , Haynes Laura , Cooper Andrea M. , Appelberg Rui
Published: May 2005

Infection by virulentMycobacterium aviumcaused progressive severe lymphopenia in C57BL/6 mice due to increased apoptosis rates. T-cell depletion did not occur in gamma interferon (IFN-γ)-deficient mice which showed increased T-cell numbers and proliferation; in contrast, deficiency in nitric oxide synthase 2 did not prevent T-cell loss. Although T-cell loss was IFN-γ dependent, expression of the IFN-γ receptor on T cells was not required for depletion. Similarly, while T-cell loss was optimal if the T cells expressed IFN-γ, CD8+T-cell depletion could occur in the absence of T-cell-derived IFN-γ. Depletion did not require that the T cells be specific for mycobacterial antigen and was not affected by deficiencies in the tumor necrosis factor receptors p55 or p75, the Fas receptor (CD95), or the respiratory burst enzymes or by forced expression ofbcl-2in hematopoietic cells.