bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effect of
mutS
and
recD
Mutations on
Salmonella
Virulence
Authors: Zahrt Thomas C. , Buchmeier Nancy , Maloy Stanley 
Published: December 2019

ABSTRACT

Hybrid derivatives of closely related bacteria may be used to dissect strain-specific functions that contribute to virulence within a host. However, mismatches between DNA sequences are a potent barrier to recombination. Recipients with
mutS
and
recD
mutations overcome this barrier, allowing construction of genetic hybrids. To determine whether
Salmonella
hybrids constructed in a
mutS recD
host can be used to study virulence, we assayed the effect of
mutS
and
recD
mutations on the virulence of
Salmonella typhimurium
14028s in mice. Mutants defective in either
mutS
or
recD
do not affect the time course or the 50% lethal dose (LD
50
) of the infection. In contrast, the inactivation of both
mutS
and
recD
results in a synthetic phenotype which substantially increases the time required to cause a lethal infection without changing the LD
50
. This phenotype results from an inability of
mutS recD
double mutants to rapidly adapt to purine-limiting conditions present within macrophages. Although the disease progression is slower,
S. typhimurium mutS recD
mutants retain the ability to cause lethal infections, and, thus, hybrids constructed in
mutS recD
hosts may permit the analysis of virulence factors in a surrogate animal model.
Homotypic and Heterotypic Antibody Responses to a 56-Kilodalton Protein of
Orientia tsutsugamushi
Authors: Choi Myung-Sik , Seong Seung-Yong , Kang Jae-Seung , Kim Yun-Won , Huh Myung-Suk , Kim Ik-Sang 
Published: December 2019

ABSTRACT

We analyzed homotypic and heterotypic antibody responses to a type-specific antigen (Tsa), a 56-kDa protein of
Orientia tsutsugamushi
, by using sera from mice immunized with strains Gilliam, Karp, Kato, and Boryong. We generated a series of deletion constructs of the
tsa
gene and expressed them as MalE fusion proteins. Variable domain I (VD I) showed strong responses to homotypic antibodies. Antigenic domain II (AD II) from Boryong and Karp showed cross-reactivities to each other. VD III showed no responses to any of the antibodies. Sera from Kato-immunized mice showed only homotypic responses to AD III. On the other hand, sera of the mice immunized with Gilliam, Karp, or Boryong showed homotypic as well as heterotypic responses to this region. VD IV showed the strongest heterotypic antibody responses among the fragments tested. These data suggest that VD I is important in homotypic antibody responses and that AD II, AD III, and VD IV are important in heterotypic antibody responses of the mice to Tsa.
P48 Major Surface Antigen of
Mycoplasma agalactiae
Is Homologous to a
malp
Product of
Mycoplasma fermentans
and Belongs to a Selected Family of Bacterial Lipoproteins
Authors: Rosati Sergio , Pozzi Sarah , Robino Patrizia , Montinaro Barbara , Conti Amedeo , Fadda Manlio , Pittau Marco 
Published: December 2019

ABSTRACT

A major surface antigenic lipoprotein of
Mycoplasma agalactiae
, promptly recognized by the host's immune system, was characterized. The mature product, P48, showed significant similarity and shared conserved amino acid motifs with lipoproteins or predicted lipoproteins from
Mycoplasma fermentans
,
Mycoplasma hyorhinis
, relapsing fever
Borrelia
spp.,
Bacillus subtilis
, and
Treponema pallidum
.
Genetic and Physiologic Characterization of Urease of
Actinomyces naeslundii
Authors: Morou-Bermudez Evangelia , Burne Robert A. 
Published: December 2019

ABSTRACT

Ammonia production from urea by ureolytic oral bacteria is believed to have a significant impact on oral health and the ecological balance of oral microbial populations. In this study we cloned and characterized the urease gene cluster of
Actinomyces naeslundii
, which is one of the pioneer organisms in the oral cavity and a significant constituent of supragingival and subgingival dental plaque in children and adults. An internal fragment of the
ureC
gene of
A. naeslundii
WVU45 was initially amplified by PCR with degenerate primers derived from conserved amino acid sequences of the large catalytic subunit of urease in bacteria and plants. The PCR product was then used as a probe to identify recombinant bacteriophages carrying the
A. naeslundii
urease gene cluster and roughly 30 kbp of flanking DNA. Nucleotide sequence analysis demonstrated that the gene cluster was comprised of seven contiguously arranged open reading frames with significant homologies at the protein and nucleotide sequence levels to the
ureABCEFGD
genes from other organisms. By using primer extension, a putative transcription initiation site was mapped at 66 bases 5′ to the start codon of
ureA
. A urease-deficient strain was constructed by insertion of a kanamycin resistance determinant within the
ureC
gene via allelic replacement. In contrast to the wild-type organism, the isogenic mutant was unable to grow in a semidefined medium supplemented with urea as the nitrogen source and was not protected by the addition of urea against killing in moderately acidic environments. These data indicated that urea can be effectively utilized as a nitrogen source by
A. naeslundii
via a urease-dependent pathway and that ureolysis can protect
A. naeslundii
against environmental acidification at physiologically relevant pH values. Therefore, urease could confer to
A. naeslundii
critical selective advantages over nonureolytic organisms in dental plaque, constituting an important determinant of plaque ecology.
Consequences of Reduction of
Klebsiella pneumoniae
Capsule Expression on Interactions of This Bacterium with Epithelial Cells
Authors: Favre-Bonte Sabine , Joly Bernard , Forestier Christiane 
Published: December 2019

ABSTRACT

Most
Klebsiella pneumoniae
clinical isolates are fully encapsulated and adhere in vitro to intestinal cell lines with an aggregative pattern. In this study, the influence of the capsule on interactions with epithelial cells was investigated by creating an isogenic mutant defective in the synthesis of the capsule. Determination of the uronic acid content of bacterial extracts confirmed that the mutant did not produce capsular polysaccharides whereas, with the wild-type strain, the level of encapsulation was growth phase dependent and reached a maximum during the lag and early log phases. Assays performed with different epithelial cell lines, Int-407, A-549, and HEp-2, showed that the capsule-defective mutant demonstrated greater adhesion than did the wild-type strain and that the aggregative pattern was maintained, indicating that the capsule was not related to the adhesion phenotype. In contrast, when the mucus-producing HT-29-MTX cells were used, the encapsulated wild-type strain adhered more strongly than did the capsule-defective mutant. No invasion properties were observed with any of the capsular phenotypes or cell lines used. The
K. pneumoniae
adhesin CF29K was detected by Western blot analysis and enzyme-linked immunosorbent assay on the surface of transconjugants obtained after transfer of a conjugative plasmid harboring the CF29K-encoding genes into both the wild-type and the capsule-defective strains. The amounts of adhesin detected were greater in the capsule-defective background strain than in the wild-type encapsulated strain and were associated with an increase in the level of adhesion to Caco-2 cells. Moreover, RNA slot blot experiments showed that transcription of the adhesin-encoding gene was markedly increased in the capsule-defective mutant compared to the wild-type encapsulated background. These results suggest (i) that the capsule plays an active role during the initial steps of the pathogenesis by interacting with mucus-producing cells but is subsequently not required for the adhesin-related interaction with the epithelial cell surface and (ii) that the expression of the adhesin is modulated by the presence of a capsule at a transcriptional level.
Binding and Utilization of Human Transferrin by
Prevotella nigrescens
Authors: Duchesne Pascale , Grenier Daniel , Mayrand Denis 
Published: December 2019

ABSTRACT

To survive and multiply within their hosts, pathogens must possess efficient iron-scavenging mechanisms. In the present study, we investigate the capacity of
Prevotella nigrescens
and
Prevotella intermedia
to use various sources of iron for growth and characterize the transferrin-binding activity of
P. nigrescens
. Iron-saturated human transferrin and lactoferrin, but not ferric chloride and the iron-free form of transferrin, could be used as sources of iron by
P. nigrescens
and
P. intermedia
. Neither siderophore activity nor ferric reductase activity could be detected in
P. nigrescens
and
P. intermedia
. However, both species showed transferrin-binding activity as well as the capacity to proteolytically cleave transferrin. To various extents, all strains of
P. nigrescens
and
P. intermedia
tested demonstrated transferrin-binding activity. The activity was heat and protease sensitive. The capacity of
P. nigrescens
to bind transferrin was decreased when cells were grown in the presence of hemin. Preincubation of bacterial cells with hemin, hemoglobin, lactoferrin, fibrinogen, immunoglobulin G, or laminin did not affect transferrin-binding activity. The transferrin-binding protein could be extracted from the cell surface of
P. nigrescens
by treatment with a zwitterionic detergent. Subjecting the cell surface extract to affinity chromatography on an agarose-transferrin column revealed that it contained a protein having an estimated molecular mass of 37 kDa and possessing transferrin-binding activity. The transferrin-binding activity of
P. nigrescens
and
P. intermedia
may permit the bacteria to obtain iron for survival and growth in periodontal pockets.
Pathophysiology of Antigen 85 in Patients with Active Tuberculosis: Antigen 85 Circulates as Complexes with Fibronectin and Immunoglobulin G
Authors: Bentley-Hibbert Stuart I. , Quan Xin , Newman Thomas , Huygen Kris , Godfrey Henry P. 
Published: December 2019

ABSTRACTAntigen 85 (Ag85) complex proteins are major secretory products ofMycobacterium tuberculosisand induce strong cellular and humoral immune responses in infected experimental animals and human beings. We have previously shown that nanogram doses of these 30- to 32-kDa fibronectin-binding proteins inhibit local expression of delayed hypersensitivity by a T-cell fibronectin-dependent mechanism. Circulating levels of Ag85 might be expected to be elevated in patients with active tuberculosis and possibly to play a role in systemic anergy in these patients. To test this hypothesis, Ag85 was measured in serum and urine by a monoclonal antibody-based dot immunobinding assay in 56 patients and controls with known skin test reactivity. Median serum Ag85 levels were 50- to 150-fold higher in patients with active tuberculosis than in patients with activeM. avium-intracellularedisease or other nontuberculous pulmonary disease or in healthy controls (P< 0.001). The median and range of serum Ag85 in patients with active tuberculosis was not significantly different between skin test-positive and -negative subjects. Patients with activeM. aviumdisease could be distinguished from those with disease due toM. tuberculosisby monoclonal anti-Ag85 antibodies of appropriate specificities. No increases in urinary Ag85 were detected in any patient, regardless of the Ag85 level in serum. Chromatographic analysis and immunoprecipitation studies of serum revealed that Ag85 existed in the serum of these patients complexed to either fibronectin or immunoglobulin G (IgG). Uncomplexed circulating Ag85 was demonstrable in serum from fewer than 20% of patients with active tuberculosis. In patients with active tuberculosis, Ag85 is therefore likely to circulate primarily as complexes with plasma fibronectin and IgG rather than in unbound form. The existence of Ag85 complexes with plasma proteins would account for its lack of urinary clearance.
Tumor Necrosis Factor Alpha Receptor I Is Important for Survival fromStreptococcus pneumoniaeInfections
Authors: O’Brien David P. , Briles David E. , Szalai Alexander J. , Tu Anh-Hue , Sanz Inaki , Nahm Moon H. 
Published: December 2019

ABSTRACTTumor necrosis factor alpha (TNF-α) is important in resistance to various microorganisms and provides signals to the target cells through two different receptors, TNF-α receptor I (TNFRI) (p55 receptor) and TNFRII (p75 receptor). To delineate the significance of the two different signaling pathways in resisting infections with extracellular bacteria, we examined the resistance of mice toStreptococcus pneumoniae(serotype 6B). TNF-α needs to be present early in infections, since one injection of wild-type mice with anti-TNF-α leads to an increased susceptibility of these mice toS. pneumoniae. TNF-α signaling through the p55 receptor (but not the p75 receptor) is crucial in resistingS. pneumoniaeinfections, because intraperitoneal injection of 100 CFU/mouse killed p55-deficient mice by day 2 of infection, whereas 1,000,000 CFU/mouse was needed to kill half of the control mice. p55-deficient mice do not show evidence of a deficient acute-phase response. All three types of mice (p55 deficient, p75 deficient, and normal) showed comparable rises in the levels of two acute-phase proteins (serum amyloid P and C3) at 24, 48, and 72 h after the experimental infections, and all of the mice showed comparable influxes of neutrophils to the site of infection. Finally, it was demonstrated that p55-deficient mice can be protected from the lethal effects ofS. pneumoniaeinfection by injection of antibodies specific forS. pneumoniaepolysaccharide capsule.
Orchestration of Neutrophil Movement by Intestinal Epithelial Cells in Response toSalmonella typhimuriumCan Be Uncoupled from Bacterial Internalization
Authors: Gewirtz Andrew T. , Siber Andrew M. , Madara James L. , McCormick Beth A. 
Published: December 2019

ABSTRACTIntestinal epithelial cells respond toSalmonella typhimuriumby internalizing this pathogen and secreting, in a polarized manner, an array of chemokines which direct polymorphonuclear leukocyte (PMN) movement. Notably, interleukin-8 (IL-8) is secreted basolaterally and directs PMN through the lamina propria, whereas pathogen-elicited epithelial chemoattractant (PEEC) is secreted apically and directs PMN migration across the epithelial monolayer to the intestinal lumen. While most studies ofS. typhimuriumpathogenicity have focused on the mechanism by which this bacterium invades its host, the enteritis characteristically associated with salmonellosis appears to be more directly attributable to the PMN movement that occurs in response to this pathogen. Therefore, we sought to better understand the relationship betweenS. typhimuriuminvasion and epithelial promotion of PMN movement. First, we investigated whetherS. typhimuriumbecoming intracellular was necessary or sufficient to induce epithelial promotion of PMN movement. BlockingS. typhimuriuminvasion by preventing, with cytochalasin D, the epithelial cytoskeletal rearrangements which mediate internalization did not reduce the epithelial promotion of PMN movement. Conversely, bacterial attainment of an intracellular position was not sufficient to induce model epithelia to direct PMN transmigration, since neither basolateral invasion byS. typhimuriumnor apical internalization of an invasion-deficient mutant (achieved by inducing membrane ruffling with epidermal growth factor) induced this epithelial cell response. These results indicate that specific interactions between the apical surface of epithelial cells andS. typhimurium, rather than simply bacterial invasion, mediate the epithelial direction of PMN transmigration. To further investigate the means by whichS. typhimuriuminduces epithelia to direct PMN movement, we investigated whether the same signaling pathways regulate secretion of IL-8 and PEEC. IL-8 secretion, but not PEEC secretion, was activated by phorbol myristate acetate and blocked by an inhibitor (mg-132) of the proteosome which mediates NF-κβ activation. Further, secretion of IL-8, but not PEEC, was activated by an entry-deficient (HilΔ)S. typhimuriummutant or by basolateral invasion of a wild-type strain. Together, these results indicate that distinct signaling pathways mediateS. typhimuriuminvasion, induction of IL-8 secretion, and induction of PEEC secretion in model intestinal epithelia.
Genetic Vaccination againstCoccidioides immitis: Comparison of Vaccine Efficacy of Recombinant Antigen 2 and Antigen 2 cDNA
Authors: Jiang Chengyong , Magee D. Mitchell , Quitugua Teresa N. , Cox Rebecca A. 
Published: December 2019

ABSTRACTPrevious studies from our laboratory established that C-ASWS, an alkali-soluble, water-soluble extract from cell walls ofCoccidioides immitis, protects mice against lethal challenge with this fungus. The C-ASWS extract contains a glycosylated protein, designated antigen 2 (Ag2), and a polysaccharide antigen. We recently cloned Ag2 cDNA and showed that the recombinant fusion protein elicited strong delayed-type hypersensitivity responses in immunized mice. This investigation was undertaken to determine if the recombinant Ag2 protein, expressed as an Ag2-glutathioneS-transferase (GST) fusion protein, or Ag2 cDNA would protect mice against lethal challenge withC. immitis. The recombinant Ag2-GST protein protected BALB/c mice against intraperitoneal challenge with 250 arthroconidia, as assessed by a decrease in fungal CFU in tissues. The Ag2-GST-immunized mice did not show, however, an increased survival during a 30-day period postinfection. By contrast, immunization of mice with Ag2 cDNA ligated into the pVR1012 plasmid engendered protection against intraperitoneal challenge with 2,500 arthroconidia and against pulmonary challenge with 50 arthroconidia. Vaccine efficacy paralleled the development of delayed-type hypersensitivity responses toC. immitisantigen. Whereas mice vaccinated with the recombinant Ag2-GST protein did not mount footpad hypersensitivity to C-ASWS or the recombinant Ag2-GST protein, mice vaccinated with the pVR1012-Ag2 construct mounted a strong footpad hypersensitivity and their spleen cells secreted gamma interferon upon in vitro stimulation with the Ag2-containing C-ASWS extract. This is the first investigation to show that genetic immunization can protect against lethal challenge withC. immitis.
An In Vitro Tissue Culture Bilayer Model To Examine Early Events inMycobacterium tuberculosisInfection
Authors: Birkness K. A. , Deslauriers M. , Bartlett J. H. , White E. H. , King C. H. , Quinn F. D. 
Published: December 2019

ABSTRACTA tissue culture bilayer system that mimics some aspects of early alveolar infection byMycobacterium tuberculosiswas developed. This model incorporates human lung epithelial type II pneumocyte (A549) (upper chamber) and endothelial cell (lower chamber) layers separated by a microporous membrane. This construction makes it possible to observe and quantify the passage of bacteria through the two layers, to observe the interaction of the bacteria with the various cell types, and to examine the basic mechanisms of immune cell recruitment to the site of infection. After 107organisms were added to the upper chamber we microscopically observed large numbers of bacteria attached to and within the pneumocytes and we determined by viable-cell counting that a small percentage of the inoculum (0.02 to 0.43%) passed through the bilayer into the lower chamber. When peripheral blood mononuclear cells were added to the lower chamber, microscopic examination indicated a migration of the mononuclear cells through the bilayer to the apical surface, where they were seen associated with the mycobacteria on the pneumocytes. The added complexity of the bilayer system offers an opportunity to define more precisely the roles of the various lung cell types in the pathogenesis of early tuberculosis.
Induction of CD18-Mediated Passage of Neutrophils byPasteurella haemolyticain Pulmonary Bronchi and Bronchioles
Authors: Ackermann Mark R. , Brogden Kim A. , Florance Allison F. , Kehrli Marcus E. 
Published: December 2019

ABSTRACTPasteurella haemolyticais an important respiratory pathogen of cattle that incites extensive infiltrates of neutrophils into the lung. In addition to the parenchymal damage caused by factors released byP. haemolytica, neutrophils contribute to the pathologic changes in the lungs. Molecules which mediate neutrophil infiltration into the lungs duringP. haemolyticapneumonia are poorly characterized. To determine whether the CD18 family (β2-integrin) of leukocyte adhesion molecules mediates initial passage of neutrophils into the pulmonary bronchi and bronchioles of lungs infected withP. haemolytica, three Holstein calves homozygous for bovine leukocyte adhesion deficiency (BLAD) (CD18-deficient neutrophils), and three age- and breed-matched control calves (normal CD18 expression) were inoculated withP. haemolyticaA1 via a fiberoptic bronchoscope and euthanized at 2 h postinoculation. Sections of lung were stained for neutrophils, and the intensity of neutrophilic infiltration was determined by computerized image analysis. Significantly fewer (P< 0.05) neutrophils infiltrated the lumen, epithelium, and adventitia of bronchioles and bronchi in lungs of calves with BLAD compared to normal calves, which had dense infiltrates within these sites at 2 h postinoculation. The reduced infiltration in calves with BLAD occurred despite the presence of an extremely large number of neutrophils in peripheral blood that is typical for these calves. The large number of neutrophils in the blood of calves with BLAD is probably a physiologic response that can occur without microbial colonization, since one calf with BLAD that was raised under germ-free conditions had large numbers of neutrophils in the blood that were similar to those in a calf with BLAD that was raised conventionally. Neutrophil counts in the germ-free and conventionally reared calves with BLAD were much higher than those in the three normal calves raised under germ-free conditions. The work in this study demonstrates that during the initial inflammatory response, neutrophils with normal CD18 expression pass more readily than CD18-deficient neutrophils into the walls and lumen of bronchi and bronchioles. It suggests that CD18 is needed for initial passage through the extensive extracellular matrix of the bronchi and bronchioles. This has potential importance for the development of therapies to direct or inhibit neutrophil infiltration into conducting airways rather than alveolar spaces.
Bacterial Invasion Is Not Required for Activation of NF-κB in Enterocytes
Authors: Eaves-Pyles Tonyia , Szabó Csaba , Salzman Andrew L. 
Published: December 2019

ABSTRACTPathogenic enteric microorganisms induce the NF-κB-dependent expression of proinflammatory genes in intestinal epithelial cells. The purpose of the present study was to clarify the contribution of microbial invasion to the degradation of the regulatory protein IκBα and the subsequent activation of NF-κB in cultured intestinal epithelial cells. Caco-2BBe cells were incubated withSalmonella dublin,Salmonella typhimurium, or a weakly invasive strain ofE. coli.S. dublinandS. typhimurium(107organisms/ml) induced equivalent concentration-dependent gel mobility shifts of an NF-κB consensus sequence that was preceded by IκBα degradation.E. coli(107organisms/ml) did not induce IκBα degradation or NF-κB translocation. Pretreatment with cytochalasin D blocked invasion of all three strains but had no effect on IκBα degradation or NF-κB activation.S. dublinandS. typhimuriumadhered to Caco-2BBe cells 3- to 10-fold more thanE. coli. NF-κB activation was prevented by physical separation ofS. dublinfrom Caco-2BBe cells by a 0.4-μm-pore-size filter. Our results imply that bacterial adhesion, rather than invasion or release of a secreted factor, is sufficient to induce IκBα degradation and NF-κB activation in intestinal epithelial cells. Our data suggest that strategies to reduce enteric inflammation should be directed to the reduction of bacterial enterocyte adhesion.