bluuebunny/crossref_metadata_2025 huggingface dataset:
The pH of the Host Niche Controls Gene Expression in and Virulence of
Candida albicans
Authors: De Bernardis Flavia , Mühlschlegel Fritz A. , Cassone Antonio , Fonzi William A.
Published: December 2019

Little is known of the biological attributes conferring pathogenicity on the opportunistic fungal pathogen
Candida albicans
. Infection by this pathogen, as for bacterial pathogens, may rely upon environmental signals within the host niche to regulate the expression of virulence determinants. To determine if
C. albicans
responds to the pH of the host niche, we tested the virulence of strains with mutations in either of two pH-regulated genes,
PHR1
and
PHR2
. In vitro,
PHR1
is expressed when the ambient pH is at 5.5 or higher and deletion of the gene results in growth and morphological defects at neutral to alkaline pHs. Conversely,
PHR2
is expressed at an ambient pH below 5.5, and the growth and morphology of the null mutant is compromised below this pH. A
PHR1
null mutant was avirulent in a mouse model of systemic infection but uncompromised in its ability to cause vaginal infection in rats. Since systemic pH is near neutrality and vaginal pH is around 4.5, the virulence phenotype paralleled the pH dependence of the in vitro phenotypes. The virulence phenotype of a
PHR2
null mutant was the inverse. The mutant was virulent in a systemic-infection model but avirulent in a vaginal-infection model. Heterozygous mutants exhibited partial reductions in their pathogenic potential, suggesting a gene dosage effect. Unexpectedly, deletion of
PHR2
did not prevent hyphal development in vaginal tissue, suggesting that it is not essential for hyphal development in this host niche. The results suggest that the pH of the infection site regulates the expression of genes essential to survival within that niche. This implies that the study of environmentally regulated genes may provide a rationale for understanding the pathobiology of
C. albicans
.
Cloning and Mutagenesis of a Serotype-Specific DNA Region Involved in Encapsulation and Virulence ofActinobacillus pleuropneumoniaeSerotype 5a: Concomitant Expression of Serotype 5a and 1 Capsular Polysaccharides in RecombinantA. pleuropneumoniaeSerotype 1
Authors: Ward Christine K. , Lawrence Mark L. , Veit Hugo P. , Inzana Thomas J.
Published: December 2019

A DNA region involved inActinobacillus pleuropneumoniaeserotype 5 capsular polysaccharide (CP) biosynthesis was identified and characterized by using a probe specific for thecpxDgene involved in CP export. The adjacent serotype 5-specific CP biosynthesis region was cloned from a 5.8-kbBamHI fragment and an 8.0-kbEcoRI fragment of strain J45 genomic DNA. DNA sequence analysis demonstrated that this region contained four complete open reading frames,cps5A,cps5B,cps5C, andcps5D. Cps5A, Cps5B, and Cps5C showed low homology with several bacterial glycosyltransferases involved in the biosynthesis of lipopolysaccharide or CP. However, Cps5D had high homology with KdsA proteins (3-deoxy-d-manno-2-octulosonic acid 8-phosphate synthetase) from other gram-negative bacteria. The G+C content ofcps5ABCwas substantially lower (28%) than that ofcps5Dand the rest of theA. pleuropneumoniaechromosome (42%). A 2.1-kb deletion spanning the clonedcps5ABCopen reading frames was constructed and transferred into the J45 chromosome by homologous recombination with a kanamycin resistance cassette to produce mutant J45-100. Multiplex PCR confirmed the deletion in this region of J45-100 DNA. J45-100 did not produce intracellular or extracellular CP, indicating thatcps5A,cps5B, and/orcps5Cwere involved in CP biosynthesis. However, biosynthesis of the Apx toxins, lipopolysaccharide, and membrane proteins was unaffected by the mutation. Besides lack of CP biosynthesis, and in contrast to J45, J45-100 grew faster, was sensitive to killing in precolostral calf serum, and was avirulent in pigs at an intratracheal challenge dose three times the 50% lethal dose (LD50) of strain J45. At six times the J45 LD50, J45-100 caused mild to moderate lung lesions but not death. Electroporation ofcps5ABCintoA. pleuropneumoniaeserotype 1 strain 4074 generated strain 4074(pJMLCPS5), which expressed both serotype 1 and serotype 5 CP. However, serotype 1 capsule expression was diminished in 4074(pJMLCPS5) in comparison to 4074. The recombinant strain produced significantly less total CP (serotypes 1 and 5 CP combined) in log phase (P= 0.0012) but significantly more total CP in late stationary phase than 4074 (P< 0.0001). In addition, strain 4074(pJMLCPS5) caused less mortality and bacteremia in pigs and mice following respiratory challenge than strain 4074, indicating that virulence was affected by diminished capsule production. These results emphasize the importance of CP in the serum resistance and virulence ofA. pleuropneumoniae.
Cytotoxicity of Hemolytic, Cytotoxic Necrotizing Factor 1-Positive and -Negative
Escherichia coli
to Human T24 Bladder Cells
Authors: Island Michael D. , Cui Xiaoling , Foxman Betsy , Marrs Carl F. , Stamm Walter E. , Stapleton Ann E. , Warren John W.
Published: December 2019

Approximately one-half of
Escherichia coli
isolates from patients with cystitis or pyelonephritis produce the pore-forming cytotoxin hemolysin, a molecule with the capacity to lyse erythrocytes and a range of nucleated cell types. A second toxin, cytotoxic necrotizing factor 1 (CNF1), is found in approximately 70% of hemolytic, but rarely in nonhemolytic, isolates. To evaluate the potential interplay of these two toxins, we used epidemiological and molecular biologic techniques to compare the cytotoxicity of hemolytic, CNF1
+
, and CNF1
−
cystitis strains toward human T24 bladder epithelial cells in vitro. A total of 29 isolates from two collections of cystitis-associated
E. coli
were evaluated by using methylene blue staining of bladder monolayers at 1-h intervals after inoculation with each strain. Most (20 of 29) isolates damaged or destroyed the T24 monolayer (less than 50% remaining) within 4 h after inoculation. As a group, CNF1
+
isolates from one collection (11 strains) were less cytotoxic at 4 h than the CNF1
−
strains in that collection (
P
= 0.009), but this pattern was not observed among isolates from the second collection (18 strains). To directly evaluate the role of CNF1 in cytotoxicity of hemolytic
E. coli
without the variables present in multiple clinical isolates, we constructed mutants defective in production of CNF1. Compared to the CNF1
+
parental isolates, no change in cytotoxicity was detected in these
cnf1
mutants. Our results indicate that CNF1 does not have a detectable effect on the ability of hemolytic
E. coli
to damage human bladder cell monolayers in vitro.
Monocytic Differentiation Inhibits Infection and Granulocytic Differentiation Potentiates Infection by the Agent of Human Granulocytic Ehrlichiosis
Authors: Klein Marina B. , Hayes Stanley F. , Goodman Jesse L.
Published: December 2019

Human granulocytic ehrlichiosis (HGE) is an emerging tick-borne infection with a specific tropism for granulocytes. We previously isolated and cultivated the HGE agent in the promyelocytic leukemia cell line HL-60 and have also demonstrated the susceptibility of both granulocytic and monocytic human marrow progenitors. Circulating monocytes have not been observed to be infected, suggesting that cell susceptibility may be differentiation specific. To evaluate this hypothesis, HL-60 cells were differentiated towards granulocytes (with dimethyl sulfoxide or all-
trans
retinoic acid) or toward monocytes-macrophages (with 12-
O
-tetradecanoylphorbol-13-acetate [TPA], gamma interferon, or 1,25-dihydroxyvitamin D
3
) and then challenged with HGE. HGE binding, internalization, and proliferation were compared in differentiated and untreated control HL-60 cells by immunofluorescence, electron microscopy, and Giemsa staining. Granulocytic differentiation resulted in a doubling of HGE binding and enhanced infection consistent with the agent’s clinical tropism for neutrophils. Granulocytic cells were unable to kill internalized ehrlichiae even after activation induced by
N
-formyl-Met-Leu-Phe alone or together with tumor necrosis factor alpha. In contrast, monocyte-macrophage differentiation with TPA resulted in complete resistance to infection through at least two distinct mechanisms: (i) reduction in binding and uptake and (ii) killing of any internalized organisms. Diminished binding in TPA-treated cells correlated with their reduced expression of sialyl Lewis x (CD15s), a putative cellular receptor component for HGE. The degree of monocytic differentiation and activation induced (i.e., TPA > gamma interferon > vitamin D
3
) correlated with resistance to HGE. Thus, HL-60 cells exhibit a striking differentiation-specific susceptibility to HGE. Differentiation-induced changes in bacterial adhesion and killing capacity underlie the tropism of HGE for granulocytic HL-60 cells and, conversely, the resistance of activated macrophages to infection.
Attachment of
Cryptosporidium parvum
Sporozoites to Human Intestinal Epithelial Cells
Authors: Joe Angela , Verdon Renaud , Tzipori Saul , Keusch Gerald T. , Ward Honorine D.
Published: December 2019

An enzyme-linked immunosorbent assay-based attachment model using the human intestinal cell line Caco-2A was developed to study attachment of
Cryptosporidium parvum
sporozoites in vitro and to assess potential inhibitors of sporozoite binding. In this system, attachment was related to sporozoite dose, incubation time, and host cell differentiation status. Polyclonal antibodies to
C. parvum
as well as glycoprotein inhibitors of a sporozoite lectin reduced attachment. This model will be a valuable tool in elucidating specific molecules and mechanisms involved in sporozoite-host cell attachment.
Tube Phagocytosis, a Novel Way for Neutrophils To Phagocytize
Borrelia burgdorferi
Authors: Suhonen Juha , Hartiala Kaija , Viljanen Matti K.
Published: December 2019

Interactions between human neutrophils and
Borrelia burgdorferi
, the Lyme disease spirochete, were studied by dark-field microscopy combined with video technology. A previously unrecognized mechanism for neutrophils to phagocytize the spirochete was discovered. During phagocytosis, the spirochete attaches to the neutrophil head-on, the neutrophil forms a thin tubelike protrusion around the bacterium, and the fully covered spirochete is drawn into the cell.
Susceptibility of Epithelial Cells to
Pseudomonas aeruginosa
Invasion and Cytotoxicity Is Upregulated by Hepatocyte Growth Factor
Authors: Fleiszig Suzanne M. J. , Vallas Vicky , Jun Cindy H. , Mok Leo , Balkovetz Daniel F. , Roth Michael G. , Mostov Keith E.
Published: December 2019

Normal cell polarity protects epithelial cells against
Pseudomonas aeruginosa
invasion and cytotoxicity. Using epithelial cell clones with selective defects in sorting of membrane constituents, and using hepatocyte growth factor pretreatment, we found that polarized susceptibility to
P. aeruginosa
can be altered without disrupting tight junctions. The results also showed that cellular susceptibility factors for invasion and cytotoxicity are not the same, although both are localized to the basolateral cell surface in polarized epithelial cells.
Delayed-Type Hypersensitivity Responses to ESAT-6 and MPT64 fromMycobacterium tuberculosisin the Guinea Pig
Authors: Elhay Martin J. , Oettinger Thomas , Andersen Peter
Published: December 2019

Two antigens fromMycobacterium tuberculosis, ESAT-6 and MPT64, elicited delayed-type hypersensitivity (DTH) skin responses in outbred guinea pigs infected withM. tuberculosisby the aerosol and intravenous routes but not those sensitized withM. bovisBCG orM. avium. The DTH epitope of ESAT-6 was mapped to the C terminus. Nonresponders to the individual antigens were found, but all animals responded to a combination of ESAT-6 and MPT64 or their respective minimal target peptides. Correspondingly, these molecules could form the basis of a new skin test for tuberculosis.
Role of a Cytotoxic Enterotoxin in
Aeromonas
-Mediated Infections: Development of Transposon and Isogenic Mutants
Authors: Xu Xin-J. , Ferguson M. R. , Popov V. L. , Houston C. W. , Peterson J. W. , Chopra A. K.
Published: December 2019

Transposon and marker exchange mutagenesis were used to evaluate the role of
Aeromonas
cytotoxic enterotoxin (Act) in the pathogenesis of diarrheal diseases and deep wound infections. The transposon mutants were generated by random insertion of Tn
5-751
in the chromosomal DNA of a diarrheal isolate SSU of
Aeromonas hydrophila
. Some of the transposon mutants had dramatically reduced hemolytic and cytotoxic activities, and such mutants exhibited reduced virulence in mice compared to wild-type
Aeromonas
when injected intraperitoneally (i.p.). Southern blot data indicated that transposition in these mutants did not occur within the cytotoxic enterotoxin gene (
act
). The transcription of the
act
gene was affected drastically in the transposon mutants, as revealed by Northern blot analysis. The altered virulence of these transposon mutants was confirmed by developing isogenic mutants of the wild-type
Aeromonas
by using a suicide vector. In these mutants, the truncated
act
gene was integrated in place of a functionally active
act
gene. The culture filtrates from isogenic mutants were devoid of hemolytic, cytotoxic, and enterotoxic activities associated with Act. These filtrates caused no damage to mouse small intestinal epithelium, as determined by electron microscopy, whereas culture filtrates from wild-type
Aeromonas
caused complete destruction of the microvilli. The 50% lethal dose of these mutants in mice was 1.0 × 10
8
when injected i.p., compared to 3.0 × 10
5
for the wild-type
Aeromonas
. Reintegration of the native
act
gene in place of the truncated toxin gene in isogenic mutants resulted in complete restoration of Act’s biological activity and virulence in mice. The animals injected with a sublethal dose of wild-type
Aeromonas
or the revertant, but not the isogenic mutant, had circulating toxin-specific neutralizing antibodies. Taken together, these studies clearly established a role for Act in the pathogenesis of
Aeromonas
-mediated infections.
CD1 Presents Antigens from a Gram-Negative Bacterium,Haemophilus influenzaeType b
Authors: Fairhurst Rick M. , Wang Chun X. , Sieling Pete A. , Modlin Robert L. , Braun Jonathan
Published: December 2019

Human CD1 is a family of nonpolymorphic major histocompatibility complex class I-like molecules capable of presenting mycobacterial lipids, including lipoarabinomannan (LAM), to double-negative (DN; CD4−CD8−) as well as CD8+T cells. Structural similarities between LAM and the capsular polysaccharides of gram-negative bacteria led us to consider the latter as candidate CD1 ligands. We derived two CD1-restricted DN T-cell populations which proliferated toHaemophilus influenzaetype b (Hib) antigen. One T-cell population also proliferated to proteinase K-treated Hib antigen, suggesting that it recognized a nonpeptide. Our work thus expands the universe of T cell antigens to include nonpeptides distinct from mycobacterial lipids and suggests a potential role for CD1-restricted T cells in immunity to Hib.
Levels of Inhibitors of Tumor Necrosis Factor Alpha and Interleukin 1β in Urine and Sera of Patients with Urosepsis
Authors: Olszyna Dariusz P. , Prins Jan M. , Buis Barbara , van Deventer Sander J. H. , Speelman Peter , van der Poll Tom
Published: December 2019

The antiinflammatory cytokine response during urosepsis was determined by measurement of concentrations of soluble tumor necrosis factor receptor (sTNFR) types I and II, interleukin 1 receptor antagonist (IL-1ra), soluble IL-1 receptor type II (sIL-1RII), and interleukin 10 in sera and urine of 30 patients with culture-proven urinary tract infections before and 4, 24, 48, and 72 h after initiation of antibiotic therapy and in 20 healthy individuals. In serum, the levels of sTNFR types I and II, IL-1ra, and IL-10 were higher in patients than in controls. In urine, only sTNFR type I and II levels were elevated in patients. The ratios of concentrations of both types of sTNFR in urine to concentrations in serum were higher in patients than in controls. These findings indicate that during urosepsis, the antiinflammatory cytokine response is generated predominantly at the systemic level.
Lipopolysaccharides from
Campylobacter jejuni
O:41 Strains Associated with Guillain-Barré Syndrome Exhibit Mimicry of GM
1
Ganglioside
Authors: Prendergast Martina M. , Lastovica Albert J. , Moran Anthony P.
Published: December 2019

Three
Campylobacter jejuni
, biotype 2, serotype O:41 strains that were isolated from patients who developed Guillain-Barré syndrome (GBS) and one
C. jejuni
isolate from a patient who developed enteritis only were examined. The aim of the study was to determine the structure of the core oligosaccharide (OS) of the lipopolysaccharide (LPS) of
C. jejuni
serotype O:41, a serotype rarely associated with the development of GBS, and to determine if the LPS shares similar epitopes with any of the major human gangliosides. Electrophoretic analysis with silver staining or immunoblotting demonstrated that the strains had LPS profiles characteristic of low-molecular-weight LPS. Colorimetric analysis detected
N
-acetylneuraminic (sialic) acid in the core OSs of all the strains. Thin-layer chromatography with immunostaining showed that antisera raised against the GBS strains reacted with the GM
1
ganglioside, suggesting that
C. jejuni
serotype O:41 LPSs and the GM
1
ganglioside have similar epitopes. Furthermore, polyclonal anti-GM
1
and anti-asialoGM
1
antibodies cross-reacted with each
C. jejuni
O:41 LPS tested, suggesting that the serotype O:41 core OS has a GM
1
- and asialoGM
1
-like structure. LPSs extracted from
C. jejuni
serostrains O:2, O:3, and O:19 were also used in the study. Cholera toxin (a GM
1
ligand) and peanut agglutinin (a Galβ1–3GalNAc ligand) recognized all serotype O:41 LPSs and the serostrain O:2 LPS. Immunoadsorption results confirmed GM
1
relatedness. Moreover, the core OS was isolated from a GBS-associated
C. jejuni
O:41 LPS by gel permeation chromatography. An analysis by gas-liquid chromatography (GLC), GLC-mass spectrometry, and nuclear magnetic resonance showed the core OS of one of the
C. jejuni
O:41 GBS isolates to have a tetrasaccharide structure consistent with GM
1
mimicry.
Listeria monocytogenesStimulates Mucus Exocytosis in Cultured Human Polarized Mucosecreting Intestinal Cells through Action of Listeriolysin O
Authors: Coconnier Marie-Hélène , Dlissi Elyess , Robard Myriam , Laboisse Christian L. , Gaillard Jean-Louis , Servin Alain L.
Published: December 2019

When the intracellular pathogenListeria monocytogenesinfects cultured human mucosecreting polarized HT29-MTX cells apically, it induces the stimulation of mucus exocytosis without cell entry. Using a set of isogenic mutants and purified listeriolysin O (LLO), we identified theL. monocytogenesthiol-activated exotoxin LLO as the agonist of mucus secretion. We demonstrated that the LLO-induced mucus exocytosis did not result from the LLO membrane-damaging activity. We found that LLO-induced mucus exocytosis is an event requiring the binding of LLO to a brush border-associated receptor and membrane oligomerization of the exotoxin. By a pharmacological approach, we demonstrated that no regulatory system or intracellular transducing signal known to be involved in control of mucin exocytosis was activated by LLO. Based on the present data, the stimulatory action of LLO on mucin exocytosis could be accounted for either by an unknown signaling system which remains to be determined or by direct action of LLO with the membrane vesicle components involved in the intracellular vesicular transport of mucins.
Kinetics and In Vivo Induction of Genetic Variation of
vlsE
in
Borrelia burgdorferi
Authors: Zhang Jing-Ren , Norris Steven J.
Published: December 2019

The Lyme disease agent,
Borrelia burgdorferi
, is able to persistently infect humans and animals for months or years in the presence of an active immune response. It is not known how the organisms survive immune attack in the mammalian host.
vlsE
, a gene localized near one end of linear plasmid lp28-1 and encoding a surface-exposed lipoprotein in
B. burgdorferi
B31, was shown recently to undergo extensive genetic and antigenic variation within 28 days of initial infection in C3H/HeN mice. In this study, we examined the kinetics of
vlsE
sequence variation in C3H/HeN mice at 4, 7, 14, 21, and 28 days and at 7 and 12 months postinfection. Sequence changes were detected by PCR amplification and sequence analysis as early as 4 days postinfection and accumulated progressively in both C3H/HeN and CB-17 severe combined immunodeficient (SCID) mice throughout the course of infection. The sequence changes were consistent with sequential recombination of segments from multiple silent
vls
cassette sites into the
vlsE
expression site. No
vlsE
sequence changes were detected in organisms cultured in vitro for up to 84 days. These results indicate that
vlsE
recombination is induced by a factor(s) present in the mammalian host, independent of adaptive immune responses. The possible inducing conditions appear to be present in various tissue sites because isolates from multiple tissues showed similar degrees of sequence variation. The rate of accumulation of predicted amino acid changes was higher in the immunologically intact C3H/HeN mice than in SCID mice, a finding consistent with immune selection of VlsE variants.
Genetic Variation of the
Borrelia burgdorferi
Gene
vlsE
Involves Cassette-Specific, Segmental Gene Conversion
Authors: Zhang Jing-Ren , Norris Steven J.
Published: December 2019

The Lyme disease spirochete
Borrelia burgdorferi
possesses 15 silent
vls
cassettes and a
vls
expression site (
vlsE
) encoding a surface-exposed lipoprotein. Segments of the silent
vls
cassettes have been shown to recombine with the
vlsE
cassette region in the mammalian host, resulting in combinatorial antigenic variation. Despite promiscuous recombination within the
vlsE
cassette region, the 5′ and 3′ coding sequences of
vlsE
that flank the cassette region are not subject to sequence variation during these recombination events. The segments of the silent
vls
cassettes recombine in the
vlsE
cassette region through a unidirectional process such that the sequence and organization of the silent
vls
loci are not affected. As a result of recombination, the previously expressed segments are replaced by incoming segments and apparently degraded. These results provide evidence for a gene conversion mechanism in VlsE antigenic variation.
Activation of the Complement Classical Pathway (C1q Binding) by MesophilicAeromonas hydrophilaOuter Membrane Protein
Authors: Merino Susana , Nogueras Maria Mercedes , Aguilar Alicia , Rubires Xavier , Albertí Sebastian , Benedí Vicente Javier , Tomás Juan M.
Published: December 2019

The mechanism of killing ofAeromonas hydrophilaserum-sensitive strains in nonimmune serum by the complement classical pathway has been studied. The bacterial cell surface component that binds C1q more efficiently was identified as a major outer membrane protein of 39 kDa, presumably the porin II described by D. Jeanteur, N. Gletsu, F. Pattus, and J. T. Buckley (Mol. Microbiol. 6:3355–3363, 1992), of these microorganisms. We have demonstrated that the purified form of porin II binds C1q and activates the classical pathway in an antibody-independent manner, with the subsequent consumption of C4 and reduction of the serum total hemolytic activity. Activation of the classical pathway has been observed in human nonimmune serum and agammaglobulinemic serum (both depleted of factor D). Binding of C1q to other components of the bacterial outer membrane, in particular to rough lipopolysaccharide, could not be demonstrated. Activation of the classical pathway by this lipopolysaccharide was also much less efficient than activation by the outer membrane protein. The strains possessing O-antigen lipopolysaccharide bind less C1q than the serum-sensitive strains, because the outer membrane protein is less accessible, and are resistant to complement-mediated killing. Finally, a similar or identical outer membrane protein (presumably porin II) that binds C1q was shown to be present in strains from the most common mesophilicAeromonasO serogroups.
Vector Development for the Expression of Foreign Proteins in the Vaccine StrainBrucella abortusS19
Authors: Comerci Diego J. , Pollevick Guido D. , Vigliocco Ana M. , Frasch Alberto C. C. , Ugalde Rodolfo A.
Published: December 2019

A vector for the expression of foreign antigens in the vaccine strainBrucella abortusS19 was developed by using a DNA fragment containing the regulatory sequences and the signal peptide of theBrucella bcsp31gene. This fragment was cloned in broad-host-range plasmid pBBR4MCS, resulting in plasmid pBEV. As a reporter protein, a repetitive antigen ofTrypanosoma cruziwas used. The recombinant fusion protein is stably expressed and secreted into theBrucellaperiplasmic space, inducing a good antibody response against theT. cruziantigen. The expression of the repetitive antigen inBrucellaneither altered its growth pattern nor generated a toxic or lethal effect during experimental infection. The application of this strategy for the generation of live recombinant vaccines and the tagging ofB. abortusS19 vaccine is discussed. This is the first time that a recombinant protein has been expressed in the periplasm of brucellae.
Paromomycin and Geneticin Inhibit Intracellular
Cryptosporidium parvum
without Trafficking through the Host Cell Cytoplasm: Implications for Drug Delivery
Authors: Griffiths Jeffrey K. , Balakrishnan Ramaswamy , Widmer Giovanni , Tzipori Saul
Published: December 2019

Cryptosporidium parvum
, which causes intractable diarrhea and lethal wasting in people with AIDS, occupies an unusual intracellular but extracytoplasmic niche. No reliable therapy for cryptosporidiosis exists, though the aminoglycoside paromomycin is somewhat effective. We report that paromomycin and the related compound geneticin manifest their major in vitro anti-
C. parvum
activity against intracellular parasites via a mechanism that does not require drug trafficking through the host cell cytoplasm. We used both normal and transformed aminoglycoside-resistant Caco-2 or MDBK cells in these studies. Timed-exposure experiments demonstrated that these drugs inhibit intracellular but not extracellular parasites. Apical but not basolateral exposure of infected cells to these drugs led to very significant parasite inhibition, indicating an apical topological restriction of action. We estimated intracytoplasmic concentrations of paromomycin, using an intracellular bacterial killing assay, and found that
C. parvum
infection did not lead to increased paromomycin concentrations compared to those in uninfected cells. Global [
3
H]paromomycin uptake by Caco-2 cells was ∼200-fold higher than the estimated intracytoplasmic paromomycin concentration, suggestive of host cell vesicular uptake and concentration (as has been reported with other cell lines). However, preinfection exposure of Caco-2 cells to paromomycin did not result in subsequent inhibition of parasite development, indicating that if exogenous paromomycin enters the infected host cell vesicular compartment, it does not effectively communicate with the parasite. Thus, the apical membranes overlying the parasite and parasitophorous vacuole may be the unsuspected major route of entry for paromomycin and may be of importance in the design and discovery of novel drug therapies for the otherwise untreatable
C. parvum.
Increased Levels of Intracellular Calcium Are Not Required for the Formation of Attaching and Effacing Lesions by Enteropathogenic and Enterohemorrhagic
Escherichia coli
Authors: Bain Christopher , Keller Rogeria , Collington Georgina K. , Trabulsi Luiz R. , Knutton Stuart
Published: December 2019

Elevated concentrations of intracellular calcium ([Ca]
i
) have been implicated as an important signalling event during attaching and effacing (A/E) lesion formation by enteropathogenic
Escherichia coli
(EPEC). The highly localized nature of the cytoskeletal and cell surface alterations occurring during A/E lesion formation suggests that there should be equally localized EPEC-induced signalling events. To analyze further the calcium responses to infection of HEp-2 cells by EPEC, we employed calcium-imaging fluorescence microscopy, which allows both temporal and spatial measurements of [Ca]
i
in live cells. Using this imaging technique, not only were we unable to detect any significant elevation in [Ca]
i
at sites of A/E EPEC adhesion, but, with several different classical EPEC and enterohemorrhagic
E. coli
(EHEC) strains and three different infection procedures, each of which resulted in extensive A/E bacterial adhesion, we were unable to detect any significant alterations in [Ca]
i
in infected cells compared to uninfected cells. In addition, chelation of intracellular free calcium with bis-(aminophenoxy)-ethane-
N
,
N
,
N
′,
N
′-tetraacetic acid (BAPTA) did not, as previously reported, prevent A/E lesion formation. We conclude that increased [Ca]
i
are not required for A/E lesion formation by EPEC and EHEC.
Role of Streptococcal Pyrogenic Exotoxin B in the Mouse Model of Group A Streptococcal Infection
Authors: Kuo Chih-Feng , Wu Jiunn-Jong , Lin Kuei-Yuan , Tsai Pei-Jane , Lee Shiour-Ching , Jin Ying-Tai , Lei Huan-Yao , Lin Yee-Shin
Published: December 2019

Streptococcal pyrogenic exotoxin B (SPE B) is a cysteine protease produced by
Streptococcus pyogenes
. In this study, the differences in virulence between protease-positive clinical isolates and their protease-negative mutants were examined in a mouse model. Isogenic protease-negative mutants were constructed by homologous recombination, using integrational plasmids to disrupt the
speB
gene. These mutants caused less mortality and tissue damage than protease-positive strains when inoculated into BALB/c mice via air pouch, suggesting that SPE B cysteine protease plays an important role in the pathogenesis of
S. pyogenes
infection. Reconstitution of SPE B in the air pouches increased the mortality of mice receiving the
speB
mutant strain. Infiltrated cell numbers in the exudates from the air pouches of mice infected with SPE B-producing
S. pyogenes
were higher than those from mice infected with protease-negative mutants at 12 h. However, despite pretreatment with vinblastine to deplete neutrophils, injection of protease-positive bacteria still resulted in severe tissue injury, indicating that neutrophil infiltration may not be the major factor involved in SPE B-enhanced tissue damage. The role of SPE B was further confirmed by demonstrating that SPE B immunization of mice conferred protection from challenge with a lethal dose of protease-positive bacteria.
Fab-Independent Antiadhesion Effects of Secretory Immunoglobulin A on S-FimbriatedEscherichia coliAre Mediated by Sialyloligosaccharides
Authors: Schroten Horst , Stapper Christoph , Plogmann Ricarda , Köhler Henrik , Hacker Jörg , Hanisch Franz-Georg
Published: December 2019

S-fimbriatedEscherichia colistrains cause sepsis and meningitis in newborns and are known to recognize the carbohydrate sequence sialyl-(α2-3)-galactoside. We show that adhesion of cloned S-fimbriatedE. colito human epithelial cells is inhibited Fab independently by sialyloligosaccharides on secretory immunoglobulin A (s-IgA). This indicates an anti-infective function of s-IgA (Fc), particularly in early human milk.
Immunogenicity of Intranasally Administered Meningococcal Native Outer Membrane Vesicles in Mice
Authors: Saunders Nancy B. , Shoemaker David R. , Brandt Brenda L. , Moran E. Ellen , Larsen Thomas , Zollinger Wendell D.
Published: December 2019

Colonization of the human nasopharyngeal region by
Neisseria meningitidis
is believed to lead to natural immunity. Although the presence of bactericidal antibody in serum has been correlated with immunity to meningococcal disease, mucosal immunity at the portal of entry may also play an important role. This study was undertaken to examine in mice the possibility of safely using native outer membrane vesicles (NOMV) not exposed to detergent as an intranasal (i.n.) vaccine. The mucosal and systemic responses of mice to intranasal and intraperitoneal (i.p.) vaccination with NOMV were compared over a range of doses from 0.1 to 20 μg. Intranasal vaccination of mice with NOMV induced a strong systemic bactericidal antibody response, as well as a strong local immunoglobulin A immune response in the lung as determined by assay of lung lavage fluid by enzyme-linked immunosorbent assay and lung antibody secreting cells by enzyme-linked immunospot assay. However, 8- to 10-fold-higher doses of NOMV were required i.n. compared to i.p. to elicit an equivalent bactericidal antibody response in serum. Some NOMV vaccine was aspirated into the lungs of mice during i.n. immunization and resulted in an acute inflammatory response that peaked at 1 to 2 days postimmunization and was cleared by day 7. These results indicate that i.n. delivery of meningococcal NOMV in mice is highly effective in eliciting the production of both a mucosal immune response and a systemic bactericidal antibody response.
Glucose Stimulates Phagocytosis of UnopsonizedPseudomonas aeruginosaby Cultivated Human Alveolar Macrophages
Authors: Wong Simon Y. C. , Guerdoud Lila M. , Cantin André , Speert David P.
Published: December 2019

Glucose has previously been shown to increase the in vitro phagocytosis of unopsonizedPseudomonas aeruginosaby freshly explanted murine peritoneal macrophages (PM) and cultivated alveolar macrophages (AM). This study examined the effect of glucose on the same phagocytosis process in human AM in order to determine whether this phenomenon is conserved among species. Freshly explanted human AM phagocytosed unopsonizedP. aeruginosaat a low level (2 bacteria/macrophage/30 min), whereas mouse AM ingested a negligible number ofP. aeruginosa(0.01 bacterium/macrophage/30 min). Glucose had no effect on this or other phagocytic processes in freshly explanted mouse or human AM. However, following in vitro cultivation for 72 h, human AM phagocytosed three to four times more unopsonizedP. aeruginosathan did freshly explanted cells, but only in the presence of glucose. This glucose-inducible phagocytic response had also been observed in cultivated murine AM. Although similar increases were also detected for the phagocytosis of latex particles and complement-coated sheep erythrocytes by cultivated human AM, these processes were not glucose dependent. The lack of response to glucose in freshly explanted mouse AM was attributed to insufficient glucose transport; however, freshly explanted human AM exhibited significant facilitative glucose transport activity that was inhibitable by cytochalasin B and phloretin. Taken together, these results suggest that the process of glucose-inducible phagocytosis of unopsonizedP. aeruginosais conserved among macrophages from different species, including humans, and that AM, but not PM, required cultivation for this glucose effect to occur. Glucose transport by AM appears to be necessary but not sufficient for phagocytosis of unopsonizedP. aeruginosa.
Identification of an Intestinal Neutral Glycosphingolipid as a Phenotype-Specific Receptor for the K88ad Fimbrial Adhesin ofEscherichia coli
Authors: Grange Philippe A. , Erickson Alan K. , Levery Steven B. , Francis David H.
Published: December 2019

In this study, we identified a receptor for the K88ad fimbrial adhesin ofEscherichia coliin neutral glycosphingolipid preparations from intestinal epithelial cells of K88ad-adhesive pigs, which was absent in preparations from K88ad-nonadhesive pigs. Neither K88ab nor K88ac adhesin variants bound to this neutral glycosphingolipid. Because this receptor is an intestinal glycosphingolipid that binds K88ad adhesin, it has been designated IGLad. Carbohydrate compositional analysis of a partially purified preparation of IGLad identified galactose, glucose, andN-acetylglucosamine in a ratio of 1.5:1.0:0.5 as the major monosaccharides. Preliminary characterization experiments using lectins showed that IGLad contains the terminal glycanic structure Galβ1-4GlcNAc. Removal of terminal β-linked galactose residues from IGLad decreased the recognition of IGLad by the K88ad adhesin, indicating that terminal β-linked galactose is an essential component of the K88ad adhesin recognition site on IGLad. Studies with purified glycosphingolipid standards demonstrated that K88ad adhesin binds to neolactotetraosylceramide (nLc4Cer) (Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-1Cer), lactotriosylceramide (GlcNAcβ1-3Galβ1-4Glcβ1-1Cer) and lactotetraosylceramide (Galβ1-3GlcNAcβ1-3Galβ1-4Glcβ1-1Cer). Based on these studies, IGLad appears to be nLc4Cer.
Fimbria-Mediated Enhanced Attachment of NontypeableHaemophilus influenzaeto Respiratory Syncytial Virus-Infected Respiratory Epithelial Cells
Authors: Jiang Zili , Nagata Nobuo , Molina Edgar , Bakaletz Lauren O. , Hawkins Hal , Patel Janak A.
Published: December 2019

Respiratory syncytial virus (RSV) infection is known to predispose children to otitis media and sinusitis due to bacteria such as nontypeableHaemophilus influenzae(NTHI). In this study, we investigated the role of NTHI surface outer membrane protein P5-homologous fimbriae (P5-fimbriae) in attachment to RSV-exposed A549 epithelial cells. Analysis by fluorescence flow cytometry showed that a live P5-fimbriated NTHI strain (NTHIF+) attached to a higher proportion of RSV-exposed A549 cells than to control cells (mean, 68% for RSV versus 29% for control;P= 0.008), while attachment of the P5-fimbriae-deficient isogenic mutant strain (NTHIF−) was significantly lower than in control cells and rose only slightly following RSV exposure (mean, 17% for RSV versus 10% for control,P= 0.229). Attachment of NTHIF+did not correlate with the amount of RSV antigen expressed by A549 cells. Furthermore, paraformaldehyde-fixed NTHIF+also demonstrated an enhanced binding to RSV-exposed cells. Observations by transmission electronic microscopy showed that the mean number of bacteria attached per 100 RSV-exposed A549 cells was higher for NTHIF+than NTHIF−(99 versus 18; P < 0.001). No intracellular bacteria were identified. UV-irradiated conditioned supernatants collected from RSV-infected A549 cultures (UV-cRSV) also enhanced the attachment of NTHIF+to A549, suggesting the presence of a preformed soluble mediator(s) in UV-cRSV that enhances the expression of receptors for P5-fimbriae on A549 cells. In summary, RSV infection significantly enhances NTHI attachment to respiratory epithelial cells. P5-fimbria is the critical appendage of NTHI that participates in this attachment. In clinical settings, blocking of the P5-fimbria-mediated attachment of NTHIF+by passive or active immunity may reduce the morbidity due to NTHI during RSV infection.
Interaction of NK Lysin, a Peptide Produced by Cytolytic Lymphocytes, with Endotoxin
Authors: Andersson M. , Girard R. , Cazenave P.-A.
Published: December 2019

NK lysin is a 9-kDa polypeptide that was originally isolated from porcine intestinal tissue based on its antibacterial activity. It is produced by cytolytic lymphocytes and is cytolytic against a number of different types of tumor cells. Here we report the binding of NK lysin to lipopolysaccharide (LPS) and its anti-LPS activity. NK lysin binds to matrix-coated LPS fromEscherichia coli,Pseudomonas aeruginosa, and different strains ofSalmonella enterica. Lipid A and polymyxin B inhibited the binding, demonstrating a preferential interaction of NK lysin with the lipid part of LPS. Chromium-labeled lymphoma cells were lysed by NK lysin, and LPS dose-dependently inhibited the cytolysis at equimolar amounts. In the same manner, NK lysin inhibited certain LPS-stimulated effects on mouse bone marrow cells as well as LPS binding to mouse granulocytes. These results suggest that NK lysin may be a another natural LPS-binding protein from lymphocytes that may participate in the endogenous defense response associated with elevated concentrations of LPS.
Compensation for Decreased Expression of B7 Molecules onLeishmania infantum-Infected Canine Macrophages Results in Restoration of Parasite-Specific T-Cell Proliferation and Gamma Interferon Production
Authors: Pinelli Elena , Rutten Victor P. M. G. , Bruysters Martijn , Moore Peter F. , Ruitenberg E. Joost
Published: December 2019

Infection of humans and dogs byLeishmania infantummay result in visceral leishmaniasis, which is characterized by impaired T-cell-mediated immune responses to parasite antigens. Dogs are natural hosts ofLeishmaniaparasites and play an important role in the transmission of the parasites to humans. In an effort to characterize the immune response in dogs infected with this intracellular pathogen, we examined how infection withL. infantumaffects canine macrophages and the consequences for T-cell activation in vitro. We showed that the proliferation of T-cell lines to cognate antigen decreases to background levels when infected autologous monocyte-derived macrophages are used as antigen-presenting cells (APC). The observed reduction of antigen-specific T-cell proliferation was shown to be dependent on the parasite load and to require cell-to-cell interaction of T cells with the infected APC. In addition, we observed a decreased expression of costimulatory B7 molecules on infected monocyte-derived macrophages. The expression of other surface molecules involved in T-cell activation, such as major histocompatibility complex class I and class II, on these cells did not change upon infection, whereas the expression of intracellular adhesion molecule 1 was marginally increased. Compensation for the decreased expression of B7 molecules by the addition of B7-transfected cells resulted in the restoration of cell proliferation and gamma interferon (IFN-γ) production by aLeishmania-specific T-cell line. These results showed that for the activation of parasite-specific canine T cells producing IFN-γ, which are most likely involved in protective immunity, sufficient expression of B7 molecules on infected macrophages is required. Provision of costimulatory molecules may be an approach for immunotherapy of leishmaniaisis as well as for vaccine development.
An Extracellular Protease ofStreptococcus gordoniiHydrolyzes Type IV Collagen and Collagen Analogues
Authors: Juarez Zaira E. , Stinson Murray W.
Published: December 2019

Streptococcus gordoniiis a frequent cause of infective bacterial endocarditis, but its mechanisms of virulence are not well defined. In this study, streptococcal proteases were recovered from spent chemically defined medium (CDM) and fractionated by ammonium sulfate precipitation and by ion-exchange and gel filtration column chromatography. Three proteases were distinguished by their different solubilities in ammonium sulfate and their specificities for synthetic peptides. One of the enzymes cleaved collagen analogs Gly-Pro 4-methoxy-β-naphthylamide, 2-furanacryloyl-Leu-Gly-Pro-Ala (FALGPA), andp-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-Arg (pZ-peptide) and was released from the streptococci while complexed to peptidoglycan fragments. Treatment of this protease with mutanolysin reduced its 180- to 200-kDa mass to 98 kDa without loss of enzymatic activity. The purified protease cleaved bovine gelatin, human placental type IV collagen, and the Aα chain of fibrinogen but not albumin, fibronectin, laminin, or myosin. Enzyme activity was inhibited by phenylmethylsulfonyl fluoride, indicating that it is a serine-type protease. Maximum production of the 98-kDa protease occurred during growth ofS. gordoniiCH1 in CDM containing 0.075% total amino acids at pH 7.0 with minimal aeration. Higher initial concentrations of amino acids prevented the release of the protease without reducing cell-associated enzyme levels, and the addition of an amino acid mixture to an actively secreting culture stopped further enzyme release. The purified protease was stored frozen at −20°C for several months or heated at 50°C for 10 min without loss of activity. These data indicate thatS. gordoniiproduces an extracellular gelatinase/type IV collagenase during growth in medium containing minimal concentrations of free amino acids. Thus, the extracellular enzyme is a potential virulence factor in the amino acid-stringent, thrombotic, valvular lesions of bacterial endocarditis.
Vibrio anguillarumResistance to Rainbow Trout (Oncorhynchus mykiss) Serum: Role of O-Antigen Structure of Lipopolysaccharide
Authors: Boesen Henriette T. , Pedersen Karl , Larsen Jens L. , Koch Claus , Ellis Anthony E.
Published: December 2019

The sensitivity ofVibrio anguillarumto the bactericidal effect of rainbow trout serum was investigated with different strains of serogroups O1 and O2a, which are the most frequently found serogroups in clinical outbreaks of vibriosis. All of theV. anguillarumstrains were able to activate complement in rainbow trout serum, but smooth strains ofV. anguillarumserogroup O1 were resistant to complement-mediated killing in the absence of specific antibodies. In the case ofV. anguillarumserogroup O2a strains, 80% of the analyzed strains were resistant to rainbow trout serum even when specific antibodies were present. Analysis of the lipopolysaccharide structures of the testedV. anguillarumstrains showed a positive correlation between the O-antigen size of the lipopolysaccharide and resistance to serum killing. The classical complement pathway was responsible for the antibody-dependent serum killing of susceptibleV. anguillarumstrains. When serum-resistantV. anguillarumserogroup O2a strains were grown in glucose-enriched Lennox L broth, they produced lipopolysaccharide molecules with fewer high-molecular-weight O-antigen units than did strains grown in broth without the addition of glucose. Strains grown in glucose-enriched medium became sensitive to rainbow trout serum killing, indicating that the high-molecular-weight O-antigen side chains prevented the activated complement from damaging the bacterium.
Influence of Acute-Phase Parasite Load on Pathology, Parasitism, and Activation of the Immune System at the Late Chronic Phase of Chagas’ Disease
Authors: Marinho Claudio R. F. , D’Império Lima Maria Regina , Grisotto Marcos G. , Alvarez José M.
Published: December 2019

To obtain low and high parasite loads in the acute phase of Chagas’ disease, A/J mice were infected with 103or 105Trypanosoma cruzitrypomastigotes of the Y strain and treated on day 6 with benznidazol. One year later, chronically infected mice were screened for subpatent parasitemias, tissue pathology, and immune response. Mice infected with the high parasite inoculum showed higher levels of chronic parasitemias, heart and striated muscle inflammation, and activation of the immune system than did mice infected with the low inoculum. Concerning the activation of the immune system, the main findings for high-dose-infected mice were (i) increased numbers of splenocytes, with preferential expansion of CD8+and B220−CD5−cells, many of them bearing a macrophage phenotype; (ii) higher frequencies of B (B220+), CD4+, and CD8+large lymphocytes; (iii) a shift of CD4+cells towards a CD45RBLowphenotype; (iv) increased frequencies of both CD45RBLowand CD45RBHighlarge CD4+cells; (v) augmented numbers of total immunoglobulin (Ig)-secreting cells, with predominance of IgG2a-producing cells; and (vi) increased production of gamma interferon and interleukin 4. In addition, these mice presented lower IgM and higher IgG2a and IgG1 parasite-specific serum antibody levels. Our results indicate that the parasite load at the acute phase ofT. cruziinfection influences the activation of the immune system and development of Chagas’ disease pathology at the late chronic phase of the disease.