bluuebunny/crossref_metadata_2025 huggingface dataset:
Transcellular Passage of
Neisseria gonorrhoeae
Involves Pilus Phase Variation
Authors: Ilver Dag , Källström Helena , Normark Staffan , Jonsson Ann-Beth
Published: December 2019

Piliated and nonpiliated
Neisseria gonorrhoeae
organisms were added on top of confluent layers of HEC-1-B cells, each maintained on a microporous Transwell-COL membrane. The bacteria released into the lower chamber were characterized with respect to the following virulence determinants: pili, which mediate adherence to target host cells; PilE, the major pilus subunit protein; and PilC, which is involved in pilus biogenesis and adherence. Even if >99% of the added bacteria of
N. gonorrhoeae
MS11 were piliated, bacteria recovered on the other side of the cell layer were predominantly nonpiliated. The recovered clones still expressed unassembled PilE protein, but 50% had lost PilC production. Nonpiliated gonococci, in which the 5′ end of
pilE
had been deleted, were released in reduced numbers, and piliated
recA
bacteria added to the cell layer were not released at all, at time points when piliated
recA
+
clones were found at high numbers in the lower chamber. Our data indicate that bacteria producing unassembled PilE protein are selected for during passage through an epithelial cell layer. The finding that the
pilE
gene sequence was altered in the transmigrants suggests that pilin sequence variation is involved in the transcellular passage of
N. gonorrhoeae.
Diffusely Adhering
Escherichia coli
Strains Induce Attaching and Effacing Phenotypes and Secrete Homologs of Esp Proteins
Authors: Beinke Christina , Laarmann Sven , Wachter Clemens , Karch Helge , Greune Lilo , Schmidt M. Alexander
Published: December 2019

Recent epidemiological studies indicate that
Escherichia coli
strains which exhibit the diffuse-adherence phenotype (DAEC strains) represent a potential cause of diarrhea in infants. We investigated the interaction of DAEC strains isolated from diarrhea patients in Brazil and in Germany with epithelial cells in tissue culture. The investigated strains were identified as DAEC strains by (i) their attachment pattern, (ii) presence of genes associated with the Dr family of adhesins, and (iii) lack of genetic markers for other diarrhea-associated
E. coli
categories. Several clinical DAEC isolates were shown to secrete similar patterns of proteins into tissue culture medium. Protein secretion was found to be regulated by environmental parameters, namely, medium, temperature, pH, and iron concentration. DAEC strains secreting these proteins induced accumulation of actin and tyrosine-phosphorylated proteins at sites of bacterial attachment, leading to the formation of pedestals and/or extended surface structures. These changes were phenotypically similar to the attaching and effacing (A/E) lesions observed with enteropathogenic and some enterohemorrhagic
E. coli
strains carrying the locus of enterocyte effacement (LEE) pathogenicity island. Proteins homologous to the EspA, EspB, and EspD proteins, necessary for signal transduction events inducing A/E lesions, were identified by sequence analysis and cross-reaction of specific antibodies. However, initially nonadhering strains secreting these proteins induced signal transduction events only after prolonged infection. These results indicate that secretion of the Esp proteins alone is not sufficient for efficient signal transduction. This study further shows that some DAEC strains are likely to contain a homolog(s) of the LEE locus which may contribute to the pathogenic potential of DAEC.
Partial Purification and Characterization of Biological Effects of a Lipid Toxin Produced byMycobacterium ulcerans
Authors: George Kathleen M. , Barker Lucia P. , Welty Diane M. , Small P. L. C.
Published: December 2019

Organisms in the genusMycobacteriumcause a variety of human diseases. One member of the genus,M. ulcerans, causes a necrotizing skin disease called Buruli ulcer. Buruli ulcer is unique among mycobacterial diseases in that the organisms at the site of infection are extracellular and there is little acute inflammatory response. Previous literature reported the presence of a toxin in the culture supernatant ofM. ulceranswhich causes a cytopathic effect on the mouse fibroblast cell line L929 in which the adherent cells round up and detach from the tissue culture plate. Here we report partial purification of a lipid toxin from the culture supernatant ofM. ulceranswhich is capable of causing the cytopathic effect on L929 cells. We also show that this cytopathic effect is a result of cytoskeletal rearrangement. TheM. ulceranstoxin does not cause cell death but instead arrests cells in the G1phase of the cell cycle.
Expression and Characterization of Group AStreptococcusExtracellular Cysteine Protease Recombinant Mutant Proteins and Documentation of Seroconversion during Human Invasive Disease Episodes
Authors: Gubba Siddeswar , Low Donald E. , Musser James M.
Published: December 2019

A recent study with isogenic strains constructed by recombinant DNA strategies unambiguously documented that a highly conserved extracellular cysteine protease expressed byStreptococcus pyogenes(group AStreptococcus[GAS]) is a critical virulence factor in a mouse model of invasive disease (S. Lukomski, S. Sreevatsan, C. Amberg, W. Reichardt, M. Woischnik, A. Podbielski, and J. M. Musser, J. Clin. Invest. 99:2574–2580, 1997). To facilitate further investigations of the streptococcal cysteine protease, recombinant proteins composed of a 40-kDa zymogen containing a C192S amino acid substitution that ablates enzymatic activity, a 28-kDa mature protein with the C192S replacement, and a 12-kDa propeptide were purified fromEscherichia colicontaining His tag expression vectors. The recombinant C192S zymogen retained apparently normal structural integrity, as assessed by the ability of purified wild-type streptococcal cysteine protease to process the 40-kDa molecule to the 28-kDa mature form. All three recombinant purified proteins retained immunologic reactivity with polyclonal and monoclonal antibodies. Humans with a diverse range of invasive disease episodes (erysipelas, cellulitis, pneumonia, bacteremia, septic arthritis, streptococcal toxic shock syndrome, and necrotizing fasciitis) caused by six distinct M types of GAS seroconverted to the streptococcal cysteine protease. These results demonstrate that this GAS protein is expressed in vivo during the course of human infections and thereby provide additional evidence that the cysteine protease participates in host-pathogen interactions in some patients.
Mapping of the T-Cell Epitope in the Major 43-Kilodalton Glycoprotein ofParacoccidioides brasiliensisWhich Induces a Th-1 Response Protective against Fungal Infection in BALB/c Mice
Authors: Taborda Carlos P. , Juliano Maria A. , Puccia Rosana , Franco Marcello , Travassos Luiz R.
Published: December 2019

The 43-kDa glycoprotein ofParacoccidioides brasiliensisis the major diagnostic antigen of paracoccidioidomycosis, the prevalent systemic mycosis of Latin America. Apart from eliciting high antibody titers, gp43 is also immunodominant in delayed-type hypersensitivity reactions in infected animals and humans. The cellular immune response in mice to gp43 administered in complete Freund’s adjuvant involves CD4+Th-1 lymphocytes, secreting gamma interferon (IFN-γ) and interleukin 2 (IL-2) but not IL-4 and IL-10. The T-cell epitope of this antigen was mapped to a 15-amino-acid peptide (P10) based on lymphoproliferations with primed cells from three different haplotypes and on a computer-assisted protein analysis. The structural requirements of the T-cell epitope were determined by assaying a series of P10 analogous and truncated peptides. Only 12-mer or longer sequences were active, confirming presentation by major histocompatibility complex II. The HTLAIR inner core of P10 is the essential domain of the epitope, with various flanking regions possible. Immunization of mice with both gp43 and P10 led to vigorous protection against intratracheal challenge by virulentP. brasiliensis, with a >200-fold decrease in lung CFU and halting of dissemination to the spleen and liver. The protective effect of P10 is mainly attributed to an IFN-γ-mediated cellular immune response. Unlike gp43, which induces an antibody response compatible with both Th-1 and Th-2 activation in infected BALB/c mice, P10 does not induce a humoral response. Protection by gp43 and P10 was characterized by a few well-demarcated lung granulomas with numerous nonviable yeast forms or resolved lesions with no detectable fungal cells.
Comparison of Intranasal and Intramuscular Immunization against Human Immunodeficiency Virus Type 1 with a DNA-Monophosphoryl Lipid A Adjuvant Vaccine
Authors: Sasaki Shin , Hamajima Kenji , Fukushima Jun , Ihata Atsushi , Ishii Norihisa , Gorai Itsuo , Hirahara Fumiki , Mohri Hiroshi , Okuda Kenji
Published: December 2019

We compared immune responses to intranasal and intramuscular DNA vaccinations against human immunodeficiency virus type 1 with monophosphoryl lipid A (MPL) used as an adjuvant. Both routes of vaccination resulted in similar levels of cell-mediated immunity, but the intestinal secretory immunoglobulin A response was higher following intranasal immunization than after intramuscular immunization. MPL demonstrated its adjuvanticity in vaccination by both routes.
Transcriptional Regulation of Endothelial Cell Tissue Factor Expression during
Rickettsia rickettsii
Infection: Involvement of the Transcription Factor NF-κB
Authors: Shi Rui-Jin , Simpson-Haidaris Patricia J. , Lerner Norma B. , Marder Victor J. , Silverman David J. , Sporn Lee Ann
Published: December 2019

The vascular endothelial cell (EC) is a primary target of infection with
Rickettsia rickettsii
, the etiologic agent of Rocky Mountain spotted fever. Changes in gene transcription elicited by intracellular infection, including EC expression of the coagulation pathway initiator known as tissue factor (TF), may contribute to the vascular pathology observed during disease. Nuclear run-on analysis of uninfected and infected, cultured human endothelial cells revealed that the rate of TF mRNA transcription is enhanced more than twofold at 3 h following infection, thus coinciding with increased steady-state levels of TF mRNA. TF mRNA remained relatively unstable during infection, with a half-life of 1.6 h. The eukaryotic protein synthesis inhibitor cycloheximide did not block
R. rickettsii
-induced increase in TF mRNA levels and actually resulted in its superinduction, thus revealing that de novo synthesis of host cell protein was not prerequisite to this transcriptional response. Involvement of the transcription factor NF-κB in
R. rickettsii
-induced TF expression was demonstrated by using two unrelated inhibitors of NF-κB activation. The antioxidant pyrrolidinedithiocarbamate and the proteasome inhibitor
N
-tosyl-
l
-phenylalanine chloromethyl ketone blocked expression of TF mRNA and activity during infection. This study demonstrates that
R. rickettsii
infection results in transcriptional activation of the TF gene and that this response involves activation of the transcription factor NF-κB.
Listeria monocytogenesInvasion of Epithelial Cells Requires the MEK-1/ERK-2 Mitogen-Activated Protein Kinase Pathway
Authors: Tang Patrick , Sutherland Claire L. , Gold Michael R. , Finlay B. Brett
Published: December 2019

PD98059, a specific inhibitor of MEK-1 mitogen-activated protein (MAP) kinase kinase, blockedListeria monocytogenesinvasion into HeLa epithelial cells. The effects of PD98059 were reversible, as adherent extracellular bacteria were internalized upon removal of the drug. Previously, we reported thatL. monocytogenescould activate ERK-1 and ERK-2 MAP kinases through the action of listeriolysin O (LLO) on the host cell (P. Tang, I. Rosenshine, P. Cossart, and B. B. Finlay, Infect. Immun. 64:2359–2361, 1996). We have now found that two other MAP kinase pathways, those of p38 MAP kinase and c-Jun N-terminal kinase, are also activated by wild-typeL. monocytogenes. Mutants lacking functional LLO (hlymutants) were still invasive but only activated ERK-2 and only activated it at later (90-min) postinfection times. Two inhibitors ofL. monocytogenesinvasion, cytochalasin D, which disrupts actin polymerization, and wortmannin, which blocks phosphatidylinositol (PI) 3-kinase activity, did not block ERK-2 activation by wild-typeL. monocytogenesandhlymutants. However, genistein, an inhibitor of tyrosine kinases, and PD98059 both blocked invasion and decreased ERK-2 activation. These results suggest that MEK-1 and ERK-2 activities are essential forL. monocytogenesinvasion into host epithelial cells. This is the first report to show that a MAP kinase pathway is required for bacterial invasion.
Identification of Virulence-Associated Characteristics in Clinical Isolates of
Yersinia enterocolitica
Lacking Classical Virulence Markers
Authors: Grant Travis , Bennett-Wood Vicki , Robins-Browne Roy M.
Published: December 2019

Yersinia enterocolitica
is an important enteric pathogen which has well-defined virulence determinants that allow the bacteria to become established in their hosts and overcome host defenses. A number of strains obtained from patients with diarrhea, however, lack these genes. Accordingly, the mechanisms by which they cause disease are uncertain. Most of these isolates belong to biotype 1A. Strains of this biotype are also frequently isolated from a variety of nonclinical sources, such as food, soil, water, and healthy animals, and there is evidence that some of these strains are avirulent. In this study we investigated 111 strains of
Y. enterocolitica
biotype 1A, 79 from symptomatic humans and 32 from nonclinical sources, for virulence-associated characteristics. DNA hybridization studies showed that none of the strains carried sequences homologous with pYV, the ∼70-kb
Yersinia
virulence plasmid. Some strains hybridized with DNA probes for one of the following chromosomal virulence-associated genes:
ail
(7.2%),
myfA
(11.7%),
ystA
(0.9%), and
ystB
(85%). In addition, 33 strains (29.7%) produced an enterotoxin that was reactive in infant mice. However, the frequencies of these virulence-associated properties in clinical and nonclinical isolates were similar. Clinical isolates invaded HEp-2 cells and Chinese hamster ovary cells to a significantly greater extent than nonclinical strains (
P
≤ 0.002). In addition, clinical strains colonized the intestinal tracts of perorally inoculated mice for significantly longer periods than nonclinical isolates (
P
≤ 0.01). Light and electron microscopic examination of tissue culture cells incubated with invasive yersiniae revealed that the bacteria invaded selected cells in large numbers but spared others, suggesting that biotype-1A strains of
Y. enterocolitica
may invade cells by a novel mechanism. These results indicate that some clinical isolates of
Y. enterocolitica
which lack classical virulence markers may be able to cause disease via virulence mechanisms which differ from those previously characterized in enteropathogenic
Yersinia
species.
Characterization of a
Chlamydia psittaci
DNA Binding Protein (EUO) Synthesized during the Early and Middle Phases of the Developmental Cycle
Authors: Zhang Li , Douglas Annemarie L. , Hatch Thomas P.
Published: December 2019

The EUO gene (for early upstream open reading frame) of
Chlamydia psittaci
was previously found to be transcribed better at 1 than at 24 h postinfection. We found that the EUO gene encodes a minor protein that is expressed within 1 h of infection of host cells with
C. psittaci
6BC but that protein quantity peaks during the logarithmic growth phase of reticulate bodies (RBs), declines late in the infection (after 20 h) when RBs reorganize into elementary bodies (EBs), and is absent in infectious EBs. EUO protein lacks homology to known proteins but does contain a putative helix-turn-helix motif. We found that recombinant EUO binds to DNA in vitro with a relatively broad specificity. Using the bp −200 to +67 promoter region of the cysteine-rich envelope protein (
crp
) operon as a model, we show that EUO protein preferentially binds to AT-rich sequences and protects
crp
DNA from DNase I from approximately bp −60 to −9. We also found that native EUO protein in extracts of RBs binds to the promoter region of the
crp
operon, demonstrating that the DNA binding property of EUO protein is not an artifact of recombinant methods. Although EUO protein appears to bind to the
crp
operon with high affinity in vitro (

K
d

of about 15 nM), it is not known whether the protein binds the
crp
DNA in vivo.
Defective Nitric Oxide Effector Functions Lead to Extreme Susceptibility ofTrypanosoma cruzi-Infected Mice Deficient in Gamma Interferon Receptor or Inducible Nitric Oxide Synthase
Authors: Hölscher Christoph , Köhler Gabriele , Müller Uwe , Mossmann Horst , Schaub Günter A. , Brombacher Frank
Published: December 2019

Trypanosoma cruzi, the causative agent of Chagas’ disease, induces an innate and adaptive host immune response during the acute phase of infection. These responses were analyzed by comparing mouse lines deficient for the gamma interferon (IFN-γ) receptor (IFN-γR−/−) or deficient for inducible nitric oxide synthase (iNOS−/−). Both lines were highly susceptible, with similar and dramatically increased parasite burdens and severe histopathology and were incapable of surviving even very low doses, exhibiting similar mortality kinetics. This pathophysiological correlation has a common cause, since both mutant mouse strains were unable to respond to infection by producing nitric oxide (NO) with the consequence that mutant macrophages had impaired trypanocidal activities. These in vivo and subsequent in vitro studies further demonstrated that an IFN-γ-dependent pathway of iNOS induction is crucial for efficient NO production and mandatory for resisting acute infection withT. cruzi. Despite this defect, both mutant mouse strains had a rather normal proinflammatory cytokine response (interleukin-12 [IL-12], IFN-γ, IL-6), with the exception of an impaired tumor necrosis factor alpha and IL-1α response in IFN-γR−/−mice, demonstrating that only the latter two cytokines are dependent on IFN-γ activation. Moreover, polarization of T cells in type 1 and type 2 T-helper (Th1/Th2) and cytotoxic T (Tc1/Tc2) cells as well asT. cruzi-specific antibody responses were normal in IFN-γR−/−mice, demonstrating that IFN-γ is not necessary for the promotion of T-cell differentiation andT. cruzi-specific antibody responses.
Mouse β-Defensin 1 Is a Salt-Sensitive Antimicrobial Peptide Present in Epithelia of the Lung and Urogenital Tract
Authors: Bals Robert , Goldman Mitchell J. , Wilson James M.
Published: December 2019

One component of host defense at mucosal surfaces appears to be epithelium-derived peptides with antimicrobial activity called defensins. Human β-defensin 1 (hBD-1) represents the first member of the β-defensin family isolated from humans and has been implicated in the pathogenesis of cystic fibrosis. We describe in this report the isolation and characterization of a murine homolog of hBD-1 called mouse β-defensin 1 (mBD-1). The predicted amino acid sequence shows the hallmark features of other known epithelial β-defensins, including the ordered array of six cysteine residues. Analysis of a genomic clone of mBD-1 revealed two exons separated by a 15-kb intron. By use of fluorescence in situ hybridization, the mBD-1 gene was localized at the proximal portion of chromosome 8, the site where mouse α-defensins are found. Lysates from cells transfected with the mBD-1 cDNA showed antibacterial activity against gram-positive and gram-negative bacteria. mBD-1 transcripts were found in kidney, liver, and female reproductive organ tissues. In the airways, mBD-1 is expressed diffusely throughout the epithelial cells of the large proximal airways with less expression in the small distal airways and no expression in alveolar cells. The present study demonstrates that a β-defensin potentially homologous to human β-defensin 1 is present in the respiratory system and other mucosal surfaces in mice.
Serum Complement Sensitivity as a Key Factor in Lyme Disease Ecology
Authors: Kurtenbach Klaus , Sewell Henna-Sisko , Ogden Nick H. , Randolph Sarah E. , Nuttall Patricia A.
Published: December 2019

The sensitivity ofBorrelia burgdorferisensu lato to animal sera was analyzed. Complement-mediated borreliacidal effects were observed with particular combinations of host serum andBorreliagenospecies. The species-specific pattern of viability and/or lysis is highly consistent with the pattern of reservoir competence of hosts forB. burgdorferisensu lato, suggesting a key role of complement in the global ecology of Lyme borreliosis.
Neither Interleukin-6 nor Inducible Nitric Oxide Synthase Is Required for Clearance ofChlamydia trachomatisfrom the Murine Genital Tract Epithelium
Authors: Perry Linda L. , Feilzer Karen , Caldwell Harlan D.
Published: December 2019

Female mice bearing targeted mutations in the interleukin-6 or inducible nitric oxide synthase locus mounted effective immune responses following vaginal infection withChlamydia trachomatis. Chlamydial clearance rates, local Th1 cytokine production, and host antibody responses were similar to those of immunocompetent control mice. Therefore, neither gene product appears to be critical for the resolution of chlamydial infections of the urogenital epithelium.
A Monoclonal Antibody Generated by Antigen Inoculation via Tick Bite Is Reactive to the
Borrelia burgdorferi
Rev Protein, a Member of the 2.9 Gene Family Locus
Authors: Gilmore Robert D. , Mbow M. Lamine
Published: December 2019

Murine monoclonal antibodies directed against proteins of
Borrelia burgdorferi
B31 (low passage) were generated by the administration of antigen via the bite of borrelia-infected ticks. This strategy was employed as a mechanism to create antibodies against antigens presented by the natural route of tick transmission versus those presented by inoculation with cultured borreliae. One of the resultant antibodies reacted with a 17-kDa antigen from cultured
B. burgdorferi
, as seen by immunoblot analysis. This antibody was used to screen a
B. burgdorferi
genomic DNA lambda vector expression library, and an immunoreactive clone was isolated. DNA sequence analysis of this clone, containing a 2.7-kb insert, revealed several open reading frames. These open reading frames were found to be homologs of genes discovered as a multicopy gene family in the 297 strain of
B. burgdorferi
by Porcella et al. (S. F. Porcella, T. G. Popova, D. R. Akins, M. Li, J. D. Radolf, and M. V. Norgard, J. Bacteriol. 178:3293–3307, 1996). By selectively subcloning genes found in this insert into an
Escherichia coli
plasmid expression vector, the observation was made that the
rev
gene product was the protein reactive with the 17-kDa-specific monoclonal antibody. The
rev
gene product was found to be expressed in low-passage, but not in high-passage,
B. burgdorferi
B31. Correspondingly, the
rev
gene was not present in strain B31 genomic DNA from cultures that had been passaged >50 times. Serum samples from Lyme disease patients demonstrated an antibody response against the Rev protein. The generation of an anti-Rev response in Lyme disease patients, and in mice by tick bite inoculation, provides evidence that the Rev protein is expressed and immunogenic during the course of natural transmission and infection.
Alterations in Frequency of Interleukin-2 (IL-2)-, Gamma Interferon-, or IL-4-Secreting Splenocytes Induced byCandida albicansMannan and/or Monophosphoryl Lipid A
Authors: Li Shaokang P. , Lee Sang-il , Domer Judith E.
Published: December 2019

We have shown previously that intravenous injection ofCandida albicansmannan (MAN) into naive mice induced CD8+effector downregulatory cells and that such cells were not produced if mice were deficient in CD4+or I-A+cells during the early interval (≤30 h) following the introduction of MAN. Moreover, the nonspecific biological response modifier monophosphoryl lipid A (MPL), given in vivo or incubated with cells in vitro, can abrogate the MAN-specific immunomodulatory activity. The mechanism by which the abrogation is mediated is unknown, but it is hypothesized to involve cytokines. Therefore, we measured the number of cytokine-secreting cells for the Th1 cytokine interleukin-2 (IL-2) and the Th2 cytokine IL-4, as well as for gamma interferon (IFN-γ), in splenocyte populations from MAN and/or MPL-treated mice, using an enzyme-linked immunospot assay designed to detect individual cytokine-secreting cells (spot-forming cells [SFC]). Cytokine-secreting cells were demonstrated in cell suspensions enriched for CD4+cells, but no SFC could be demonstrated in populations enriched for CD8+cells. Both MAN and MPL, when administered to separate groups of animals, stimulated the production of increased numbers of cytokine-producing cells for each of the three cytokines tested. The response with respect to IL-4-secreting cells, however, was the most striking. Despite the fact that MAN and MPL independently caused increases in SFC to all three cytokines, when both MAN and MPL were administered to the same animal, all increases were reversed, and the numbers of SFC detected were at or below those detected in saline control animals. These data support the hypothesis that IL-4 is involved in MAN-specific immunoregulatory activities. The data also emphasize the fact that two immunomodulators, i.e., MAN and MPL, having similar effects when given in vivo independently, may be antagonistic when administered sequentially to the same animal.
The Protective Effects of Lactoferrin Feeding against Endotoxin Lethal Shock in Germfree Piglets
Authors: Lee Wang J. , Farmer Jeffrey L. , Hilty Milo , Kim Yoon B.
Published: December 2019

The unique germfree, colostrum-deprived, immunologically “virgin” piglet model was used to evaluate the ability of lactoferrin (LF) to protect against lethal shock induced by intravenously administered endotoxin. Piglets were fed LF or bovine serum albumin (BSA) prior to challenge with intravenousEscherichia colilipopolysaccharide (LPS), and temperature, clinical symptoms, and mortality were tracked for 48 h following LPS administration. Prefeeding with LF resulted in a significant decrease in piglet mortality compared to feeding with BSA (16.7 versus 73.7% mortality,P< 0.001). Protection against the LPS challenge by LF was also correlated with both resistance to induction of hypothermia by endotoxin and an overall increase in wellness, as quantified by a toxicity score developed for these studies. In vitro studies using a flow cytometric assay system demonstrated that LPS binding to porcine monocytes was inhibited by LF in a dose-dependent fashion, suggesting that the mechanism of LF action in vivo may be inhibition of LPS binding to monocytes/macrophages and, in turn, prevention of induction of monocyte/macrophage-derived inflammatory-toxic cytokines.
Adherence of
Streptococcus pneumoniae
to Respiratory Epithelial Cells Is Inhibited by Sialylated Oligosaccharides
Authors: Barthelson Roger , Mobasseri Ali , Zopf David , Simon Paul
Published: December 2019

To study carbohydrate-mediated adherence of
Streptococcus pneumoniae
to the human airway, we measured binding of live
S. pneumoniae
organisms to a cultured cell line derived from the lining of the conjunctiva and to primary monolayers of human bronchial epithelial cells in the presence and absence of oligosaccharide inhibitors. Both encapsulated and nonencapsulated strains of
S. pneumoniae
grown to mid-logarithmic phase in suspension culture adhered to cultured primary respiratory epithelial cells and the conjunctival cell line. Adherence of nine clinically prevalent
S. pneumoniae
capsular types studied was inhibited preferentially by sialylated oligosaccharides that terminate with the disaccharide NeuAcα2-3(or 6)Galβ1. Adherence of some strains also was weakly inhibited by oligosaccharides that terminate with lactosamine (Galβ1-4GlcNAcβ1). When sialylated oligosaccharides were covalently coupled to human serum albumin at a density of approximately 20 oligosaccharides per molecule of protein, the molar inhibitory potency of the oligosaccharide inhibitor was enhanced 500-fold. The above-mentioned experiments reveal a previously unreported dependence upon sialylated carbohydrate ligands for adherence of
S. pneumoniae
to human upper airway epithelial cells. Enhanced inhibitory potencies of polyvalent over monovalent forms of oligosaccharide inhibitors of adherence suggest that the putative adhesin(s) that recognizes the structure NeuAcα2-3(or 6)Galβ1 is arranged on the bacterial surface in such a manner that it may be cross-linked by oligosaccharides covalently linked to human serum albumin.
Effect of Group A Streptococcal Cysteine Protease on Invasion of Epithelial Cells
Authors: Tsai Pei-Jane , Kuo Chih-Feng , Lin Kuei-Yuan , Lin Yee-Shin , Lei Huan-Yao , Chen Fen-Fen , Wang Jen-Ren , Wu Jiunn-Jong
Published: December 2019

Cysteine protease of group A streptococci (GAS) is considered an important virulence factor. However, its role in invasiveness of GAS has not been investigated. We demonstrated in this study that two strains of protease-producing GAS had the ability to invade A-549 human respiratory epithelial cells. Isogenic protease mutants were constructed by using integrational plasmids to disrupt the
speB
gene and confirmed by Southern hybridization and Western immunoblot analyses. No extracellular protease activity was produced by the mutants. The mutants had growth rates similar to those of the wild-type strains and produced normal levels of other extracellular proteins. When invading A-549 cells, the mutants had a two- to threefold decrease in activity compared to that of the wild-type strains. The invasion activity increased when the A-549 cells were incubated with purified cysteine protease and the mutant. However, blockage of the cysteine protease with a specific cysteine protease inhibitor, E-64, decreased the invasion activity of GAS. Intracellular growth of GAS was not found in A-549 cells. The presence or absence of protease activity did not affect the adhesive ability of GAS. These results suggested that streptococcal cysteine protease can enhance the invasion ability of GAS in human respiratory epithelial cells.
Evolution of the Primary Immune Response toHistoplasma capsulatumin Murine Lung
Authors: Cain Judith A. , Deepe George S.
Published: December 2019

Histoplasma capsulatuminduces a cell-mediated immune response in the lungs and lymphoid organs of mammals. In this study, we analyzed the progression of the cytokine and inflammatory reactions in the lungs of mice infected intranasally withH. capsulatum. We measured cytokine mRNA levels and determined the inflammatory cell populations during the active phase of infection (<3 weeks). Transcription of genes encoding interleukin-2 (IL-2), IL-4, and IL-12 and gamma interferon (IFN-γ) was detectable as early as day 3 of infection, whereas a signal for IL-10 was never observed. Competitive PCR analysis demonstrated that enhanced expression of IL-12 mRNA was observed by day 3 and that expression of mRNA for IL-2 and IFN-γ progressively increased from day 5 to day 10. All levels declined by day 14. Analysis of the inflammatory response revealed an initial elevation in myeloid cells (Mac-1+) and natural killer (NK) cells followed by a rise in T cells, predominantly CD4+cells. Since IFN-γ is a key factor in host defense, we performed cytoplasmic staining to determine the cell populations that produced this cytokine. The hierarchy of synthesis was CD4+> CD8+> NK cells. Thus,H. capsulatumprovokes an orderly modulation of the inflammatory and cytokine responses in murine lungs.
Baculovirus Merozoite Surface Protein 1 C-Terminal Recombinant Antigens Are Highly Protective in a Natural Primate Model for Human
Plasmodium vivax
Malaria
Authors: Perera K. L. R. Lakshman , Handunnetti Shiroma M. , Holm Inge , Longacre Shirley , Mendis Kamini
Published: December 2019

A successful anti-blood stage malaria vaccine trial based on a leading vaccine candidate, the major merozoite surface antigen-1 (MSP1), is reported here. The trial was based on
Plasmodium cynomolgi
, which is a primate malaria parasite which is highly analogous to the human parasite
Plasmodium vivax
, in its natural host, the toque monkey,
Macaca sinica
. Two recombinant baculovirus-expressed
P. cynomolgi
MSP1 proteins, which are analogous to the 42- and 19-kDa C-terminal fragments of
P. falciparum
MSP1, were tested by immunizing three groups of three animals each with either p42, p19, or both together. The vaccines were delivered subcutaneously in three doses at 4-week intervals with complete and incomplete Freund’s adjuvants. Very high antibody titers were obtained against both vaccinating antigens as measured by enzyme-linked immunosorbent assay (10
6
and above) and against whole parasites as measured by indirect immunofluorescence assay (>10
5
), achieving, in most animals, about a 10-fold increase from the first to the last immunization. A blood stage challenge with
P. cynomolgi
parasites led, in three adjuvant-treated and three naive control animals, to blood infections which were patent for at least 44 days, reaching peak densities of 0.6 and 3.8%, respectively. In contrast, all except one of the nine animals in the three vaccinated groups were highly protected, showing either no parasitemia at all or transient parasitemias which were patent for only 1 or 2 days. When the three p19-vaccinated monkeys were rechallenged 6 months later, the protective efficacy was unchanged. The success of this trial, and striking analogies of this natural host-parasite system with human
P. vivax
malaria, suggests that it could serve as a surrogate system for the development of a human
P. vivax
malaria vaccine based on similar recombinant analogs of the
P. vivax
MSP1 antigen.
Inhibition of the Production of Anti-OspA Borreliacidal Antibody with T Cells from Hamsters Vaccinated againstBorrelia burgdorferi
Authors: Jensen Jani R. , Du Chateau Brian K. , Munson Erik L. , Callister Steven M. , Schell Ronald F.
Published: December 2019

The serious morbidity associated with Lyme borreliosis has focused considerable effort on the development of a comprehensive vaccine for protection against infection withBorrelia burgdorferi. Induction of borreliacidal antibody by vaccination or infection has been shown to correlate with protection of humans and animals against infection with the Lyme spirochete. In this report, we showed that high levels of borreliacidal antibody (titer of 1,280) were produced in vitro when T and B cells from hamsters 14 days after vaccination were incubated with macrophages andB. burgdorferi. By contrast, T and B cells from hamsters 7 or 21 days after vaccination failed to initiate production of borreliacidal activity. Furthermore, the T cells from hamsters 7 or 21 days after vaccination inhibited the in vitro production of borreliacidal antibody when cocultured with T and B cells obtained from hamsters 14 days after vaccination. When cell-free supernatants from the suspensions of T and B cells from hamsters 14 days after vaccination were absorbed with recombinant OspA, they lost nearly all borreliacidal activity. The removal of anti-OspA antibody resulted in a decrease in borreliacidal titer from 1,280 to less than 4. These results demonstrate that T cells from vaccinated animals can prevent a sustained production of protective borreliacidal antibody.
Characterization of Anticapsular Monoclonal Antibodies That Regulate Activation of the Complement System by theCryptococcus neoformansCapsule
Authors: Kozel Thomas R. , deJong Bouke C. H. , Grinsell Matthew M. , MacGill Randall S. , Wall Kevin K.
Published: December 2019

Incubation of the encapsulated yeastCryptococcus neoformansin human serum leads to alternative pathway-mediated deposition of C3 fragments in the capsule. We examined the ability of monoclonal antibodies (MAbs) specific for different epitopes of the major capsular polysaccharide to alter the kinetics for classical and alternative pathway-mediated deposition of C3 onto a serotype A strain. We studied MAbs reactive with capsular serotypes A, B, C, and D (MAb group II); serotypes A, B, and D (MAb group III); and serotypes A and D (MAb group IV). The MAb groupings are based on antibody variable region usage which determines the antibody molecular structure. When both the classical and alternative pathways were operative, group II MAbs induced early classical pathway-mediated binding of C3 but reduced the overall rate of C3 accumulation and the amount of bound C3. Group III MAbs closely mimicked the effects of group II MAbs but exhibited reduced support of early classical pathway-facilitated accumulation of C3. Depending on the antibody isotype, group IV MAbs slightly or markedly enhanced early binding of C3 but had no effect on either the rate of C3 accumulation or the amount of bound C3. When the classical pathway was blocked, group II and III MAbs markedly suppressed C3 binding that normally would have occurred via the alternative pathway. In contrast, MAbs of group IV had no effect on alternative pathway-mediated C3 binding. These results indicate that anticapsular antibodies with different epitope specificities may have distinct regulatory effects on activation and binding of C3.
Bivalency Is Required for Anticapsular Monoclonal Antibodies To Optimally Suppress Activation of the Alternative Complement Pathway by theCryptococcus neoformansCapsule
Authors: Kozel Thomas R. , MacGill Randall S. , Wall Kevin K.
Published: December 2019

Encapsulated cells ofCryptococcus neoformansare potent activators of the alternative complement pathway. Previous studies found that monoclonal antibodies (MAbs) specific for the major capsular polysaccharide, termed glucuronoxylomannan (GXM), can markedly suppress the ability of the capsule to accumulate C3 from normal human serum via the alternative pathway. The present study examined the abilities of F(ab)2and Fab fragments of three MAbs (MAbs 439, 3C2, and 471) to mediate the suppressive effect. The results showed that F(ab)2fragments of all three MAbs suppressed activation and binding of C3 via the alternative pathway in a manner similar to that of intact antibodies. In contrast, Fab fragments of MAb 439 and MAb 3C2 showed no suppressive activity, and Fab fragments of MAb 471 were markedly reduced in suppressive activity. Indeed, there was an earlier accumulation of C3 on encapsulated cryptococci in the presence of the Fab fragments. Study of subclass switch families of MAb 439 and MAb 471 found that MAbs of an immunoglobulin G (IgG) subclass with increased flexibility in the hinge region (IgG2b) had less suppressive activity than MAbs of IgG subclasses with less flexibility (IgG1 or IgG2a). Taken together, these results indicate that cross-linking of the capsular matrix is an essential component in suppression of the alternative complement pathway by anti-GXM MAbs.
Characterization of the Carbohydrate Moiety of Intestinal Mucin-Type Sialoglycoprotein Receptors for the K88ac Fimbrial Adhesin of
Escherichia coli
Authors: Grange Philippe A. , Erickson Alan K. , Anderson Timothy J. , Francis David H.
Published: December 2019

We have previously identified two mucin-type sialoglycoproteins from porcine intestinal epithelial cells with approximate molecular masses of 210 (intestinal mucin-type glycoprotein IMTGP-1) and 240 kDa (IMTGP-2) as receptors for the K88ab and K88ac fimbrial adhesins of
Escherichia coli
. These receptors are detected in intestinal brush border membrane preparations from pigs with adhesive phenotypes but not from pigs with nonadhesive phenotypes and are postulated to be important determinants of the susceptibility of pigs to K88ab
+
and K88ac
+
enterotoxigenic
E. coli
infections. Using exoglycosidase digestion studies, we have now determined that β-linked galactose is an important component in the recognition of IMTGP-1 and IMTGP-2 by the K88ac adhesin. In addition, we observed a differential distribution of the K88ac adhesin binding activity of IMTGP-1 and IMTGP-2 along the crypt-villus axis, suggesting that receptor activity is dependent on the maturation state of the intestinal epithelial cells. Brush borders from immature intestinal epithelial cells possessed the highest concentrations of IMTGP-1 and IMTGP-2 receptor activity, with a progressive decrease in receptor activity as the cells mature. To characterize the differences in the carbohydrate moieties of IMTGP-1 and IMTGP-2, we developed a procedure for purifying the receptors, using phenol extraction followed by serial lectin affinity chromatography. Carbohydrate compositional analysis of the purified receptors indicated that the carbohydrate moieties of IMTGP-1 and IMTGP-2 consist of both N- and O-glycans containing galactose, glucose, sialic acid, mannose,
N
-acetylgalactosamine,
N
-acetylglucosamine, and fucose. The major difference between the two receptors is that IMTGP-2 contains a higher percentage of monosaccharides (mannose and glucose) commonly found in N-glycans.
Expression of Multiple Pili by
Legionella pneumophila
: Identification and Characterization of a Type IV Pilin Gene and Its Role in Adherence to Mammalian and Protozoan Cells
Authors: Stone Barbara J. , Kwaik Yousef Abu
Published: December 2019

Legionella pneumophila
expresses pili of variable lengths, either long (0.8 to 1.5 μm) or short (0.1 to 0.6 μm), that can be observed by transmission electron microscopy. We have identified a gene in
L. pneumophila
with homology to the type IV pilin genes (

pilE
L

). An insertion mutation was constructed in

pilE
L

and introduced into the
L. pneumophila
wild-type strain by allelic exchange. The pilin mutant is defective for expression of long pili. Reintroduction of the pilin locus on a cosmid vector restores expression of the long pili. The

L. pneumophila pilE
L

mutant exhibited approximately a 50% decrease in adherence to human epithelial cells (HeLa and WI-26 cells), macrophages (U937 cells), and
Acanthamoeba polyphaga
but had a wild-type phenotype for intracellular replication within these cells. Southern hybridization analysis showed that the

pilE
L

locus is present in
L. pneumophila
serogroups 1 through 13 but is variable in 16 other
Legionella
species. The presence of a type IV pilin gene and its expression by
L. pneumophila
may provide an advantage for colonization of lung tissues during Legionnaires’ disease and invasion of amoebas in the environment.
Binding to Human Extracellular Matrix by
Neisseria meningitidis
Authors: Eberhard Thomas , Virkola Ritva , Korhonen Timo , Kronvall Göran , Ullberg Måns
Published: December 2019

Adhesion of
Neisseria meningitidis
strains to extracellular matrix (ECM) and purified matrix components was examined. Most strains bound to subendothelial ECM as well as to immobilized fibronectin and types I, III, and V collagen. Strains from healthy carriers adhered significantly better than isolates from patients. The binding site was localized to the central 75-kDa cell-binding domain of the fibronectin molecule. This domain has not been described previously to interact with bacterial structures.
Increased Leptin Expression in Mice with Bacterial Peritonitis is Partially Regulated by Tumor Necrosis Factor Alpha
Authors: Moshyedi Armin K. , Josephs Michael D. , Abdalla Eddie K. , MacKay Sally L. D. , Edwards Carl K. , Copeland Edward M. , Moldawer Lyle L.
Published: December 2019

Plasma leptin and
ob
gene mRNA levels were increased in mice following bacterial peritonitis, and blocking an endogenous tumor necrosis factor alpha (TNF-α) response blunted the increase. However, plasma leptin concentrations did not correlate with the associated anorexia. We conclude that leptin expression is under partial regulatory control of TNF-α in peritonitis, but the anorexia is not dependent on increased leptin production.
Cloning and Sequencing of a
Candida albicans
Catalase Gene and Effects of Disruption of This Gene
Authors: Wysong Deborah R. , Christin Laurent , Sugar Alan M. , Robbins Phillips W. , Diamond Richard D.
Published: December 2019

Catalase plays a key role as an antioxidant, protecting aerobic organisms from the toxic effects of hydrogen peroxide, and in some cases has been postulated to be a virulence factor. To help elucidate the function of catalase in
Candida albicans
, a single
C. albicans
-derived catalase gene, designated
CAT1
, was isolated and cloned. Degenerate PCR primers based on highly conserved areas of other fungal catalase genes were used to amplify a 411-bp product from genomic DNA of
C. albicans
ATCC 10261. By using this product as a probe, catalase clones were isolated from genomic libraries of
C. albicans
. Nucleotide sequence analysis revealed an open reading frame encoding a protein of 487 amino acid residues. Construction of a
CAT1
-deficient mutant was achieved by using the Ura-blaster technique for sequential disruption of multiple alleles by integrative transformation using
URA3
as a selectable marker. Resulting mutants exhibited normal morphology and comparable growth rates of both yeast and mycelial forms. Enzymatic analysis revealed an abundance of catalase in the wild-type strain but decreasing catalase activity in heterozygous mutants and no detectable catalase in a homozygous null mutant. In vitro assays showed the mutant strains to be more sensitive to damage by both neutrophils and concentrations of exogenous peroxide that were sublethal for the parental strain. Compared to the parental strain, the homozygous null mutant strain was far less virulent for mice in an intravenous infection model of disseminated candidiasis. Definitive linkage of
CAT1
with virulence would require restoration of activity by reintroduction of the gene into mutants. However, initial results in mice, taken together with the enhanced susceptibility of catalase-deficient hyphae to damage by human neutrophils, suggest that catalase may enhance the pathogenicity of
C. albicans.
Nonspecific Binding ofClostridium difficileToxin A to Murine Immunoglobulins Occurs via the Fab Component
Authors: Cooke Deborah L. , Borriello S. Peter
Published: December 2019

Clostridium difficiletoxin A binds nonspecifically to a mouse monoclonal antibody (MAb) immunoglobulin G3 λ chain [IgG3(λ)], through the Fab component. This binding, which is retained even after boiling the MAb, is temperature dependent, with more toxin bound at 4 than 37°C (P= 0.0024). The nonspecific binding was decreased by incubation of the IgG3 λ MAb with α- or β-galactosidase (P= 0.0001 and 0.029, respectively), indicating that toxin A binds to a carbohydrate moiety on the Fab. However, binding was not blocked by theBandeiraea simplicifolialectin BS-1, indicating that a terminal α-galactose may not be involved. Binding was also not affected by competitive assays with Lewis X antigen. The dependence on carbohydrate moieties in nonspecific binding was also shown for two other MAbs, IgA(κ) and IgM(λ), with demonstration of a significant reduction in binding with α-galactosidase (P= 0.0001 and 0.0002, respectively) but not β-galactosidase (P= 0.27 and 0.25, respectively).