bluuebunny/crossref_metadata_2025 huggingface dataset:
Stimulation of Nitric Oxide Production in Macrophages byBabesia bovis
Authors: Stich Roger W. , Shoda Lisl K. M. , Dreewes Miriam , Adler Barbara , Jungi Thomas W. , Brown Wendy C.
Published: March 2020

Gamma interferon (IFN-γ)-activated macrophages are believed to play a key role in resistance toBabesia bovisthrough parasite suppression by macrophage secretory products. However, relatively little is known about interactions between this intraerythrocytic parasite and the macrophages of its bovine host. In this study, we examined the in vitro effect of intact and fractionatedB. bovismerozoites on bovine macrophage nitric oxide (NO) production. In the presence of IFN-γ,B. bovismerozoites stimulated NO production, as indicated by the presence of increasedl-arginine-dependent nitrite (NO2−) levels in culture supernatants of macrophages isolated from several cattle. The merozoite crude membrane (CM) fraction stimulated greater production of NO, in a dose-dependent manner, than did the merozoite homogenate or the soluble, cytosolic high-speed supernatant fraction. Stimulation of NO production by CM was enhanced by as little as 1 U of IFN-γ per ml of culture medium. Upregulation of inducible NO synthase mRNA in bovine macrophages by eitherB. bovis-parasitized erythrocytes and IFN-γ or CM was also observed.B. bovis-specific T-helper lymphocyte culture supernatants, all of which contained IFN-γ, were also found to inducel-arginine-dependent NO2−production. Supernatants that induced the highest levels of NO also contained biologically active TNF. These results show thatB. bovismerozoites and antigen-stimulatedB. bovis-immune T cells can induce the production of NO, a molecule implicated in both protection and pathologic changes associated with hemoprotozoan parasite infections.
Production and Characterization of a Set of Mouse-Human Chimeric Immunoglobulin G (IgG) Subclass and IgA Monoclonal Antibodies with Identical Variable Regions Specific forPseudomonas aeruginosaSerogroup O6 Lipopolysaccharide
Authors: Preston Michael J. , Gerçeker A. Alev , Reff Mitchell E. , Pier Gerald B.
Published: March 2020

The heavy- and light-chain variable regions from a murine monoclonal antibody that recognizePseudomonas aeruginosaserogroup O6 lipopolysaccharide (LPS) were used to generate a series of chimeric mouse-human monoclonal antibodies with identical variable regions. The murine variable-region gene segments were cloned into an immunoglobulin (Ig) cDNA expression vector that contained the human kappa light-chain and IgG1 constant regions. The IgG1 heavy-chain constant region was then replaced with the human IgG2, IgG3, IgG4, or IgA1 heavy-chain constant region. The five different expression vectors were transfected into Chinese hamster ovary cells for antibody production. The chimeric antibodies exhibited immunoreactivity and affinity similar to that of the parental murine IgG antibody toward whole cells of a serogroup O6 strain. In vitro complement deposition assays demonstrated that the chimeric IgG4 and IgA antibodies did not mediate the deposition of complement component C3 onto the surface of either purified LPS or whole bacteria. The chimeric IgG1 and IgG3 antibodies were similar in their ability to deposit C3 onto the surface of both bacteria and LPS, while IgG2 antibody was more effective at depositing C3 onto the surface of bacteria than onto purified LPS. The pattern of opsonophagocytic activity of the chimeric monoclonal antibodies was similar to that of complement deposition onto bacterial cells in that the chimeric IgG1 and IgG3 had the highest opsonic activity. Although IgG2 deposited more C3 onto the bacterial surface than did IgG4 or IgA, all three of these isotypes had low opsonic activity against the serogroup O6 target strain. This series of related antibodies will help reveal functional differences in efficacy among protective antibodies toP. aeruginosaand will be critical for defining the optimal formulation of either a vaccine for active immunization or a polyclonal intravenous IgG or monoclonal antibody cocktail for passive immunotherapy.
Requirement of theShigella flexneriVirulence Plasmid in the Ability To Induce Trafficking of Neutrophils across Polarized Monolayers of the Intestinal Epithelium
Authors: McCormick Beth A. , Siber Andrew M. , Maurelli Anthony T.
Published: March 2020

Attachment of an array of enteric pathogens to epithelial surfaces is accompanied by recruitment of polymorphonuclear leukocytes (PMN) across the intestinal epithelium. In this report, we examine howShigella-intestinal epithelium interactions evoke the mucosal inflammatory response. We modeled these interactions in vitro by using polarized monolayers of the human intestinal epithelial cell line, T84, isolated human PMNs, andShigella flexneri. We show thatShigellaattachment to T84-cell basolateral membranes was a necessary component in the signaling cascade for induction of basolateral-to-apical directed transepithelial PMN migration, the direction of PMN transepithelial migration in vivo. In contrast, attachment ofShigellato the T84-cell apical membrane failed to stimulate a directed PMN transepithelial migration response. Importantly, the ability ofShigellato induce PMN migration across epithelial monolayers was dependent on the presence of the 220-kb virulence plasmid. Moreover, examination ofShigellagenes necessary to signal subepithelial neutrophils established the requirement of a functional type III secretion system. Our results indicate that the ability ofShigellato elicit transepithelial signaling to neutrophils from the basolateral membrane of epithelial cells represents a mechanism involved inShigella-elicited enteritis in humans.
The Phosphorylcholine Epitope Undergoes Phase Variation on a 43-Kilodalton Protein in
Pseudomonas aeruginosa
and on Pili of
Neisseria meningitidis
and
Neisseria gonorrhoeae
Authors: Weiser Jeffrey N. , Goldberg Joanna B. , Pan Nina , Wilson Lynn , Virji Mumtaz
Published: March 2020

Phosphorylcholine (ChoP) is a component of the teichoic acids of
Streptococcus pneumoniae
and has been recently identified on the lipopolysaccharide of
Haemophilus influenzae
, also a major pathogen of the human respiratory tract. Other gram-negative pathogens that frequently infect the human respiratory tract were surveyed for the presence of the ChoP epitope as indicated by binding to monoclonal antibodies (MAbs) recognizing this structure. The ChoP epitope was found on a 43-kDa protein on all clinical isolates of
Pseudomonas aeruginosa
examined and on several class I and II pili of
Neisseria meningitidis
. The specificity of the anti-ChoP MAb was demonstrated by the inhibition of binding in the presence of ChoP but not structural analogs. As in the case of
H. influenzae
, the expression of this epitope was phase variable on these species. In
P. aeruginosa
, this epitope was expressed at detectable levels only at lower growth temperatures. Expression of the ChoP epitope on piliated neisseriae displayed phase variation, both linked to pilus expression and independently of fully piliated bacteria.
Contribution of Regulation by the
bvg
Locus to Respiratory Infection of Mice by
Bordetella pertussis
Authors: Merkel Tod J. , Stibitz Scott , Keith Jerry M. , Leef Mary , Shahin Roberta
Published: March 2020

Whooping cough is an acute respiratory disease caused by the small, gram-negative bacterium
Bordetella pertussis. B. pertussis
expresses several factors that contribute to its ability to cause disease. These factors include surface-associated molecules, which are involved in the adherence of the organism to respiratory epithelial cells, as well as several extracellular toxins that inhibit host defenses and induce damage to host tissues. The expression of virulence factors in
B. pertussis
is dependent upon the
bvg
locus, which consists of three genes:
bvgA
,
bvgS
, and
bvgR
. The
bvgAS
genes encode a two-component regulatory system consisting of a sensor protein, BvgS, and a transcriptional activator, BvgA. Upon modification by BvgS, BvgA binds to the promoter regions of the
bvg
-activated genes and activates transcription. One of the
bvg
-activated genes,
bvgR
, is responsible for the regulation of the
bvg
-repressed genes, the functions of which are unknown. The fact that these genes are regulated by the
bvg
locus suggests that they play a role in the pathogenesis of the bacterium. In order to evaluate the contribution of
bvg
-mediated regulation to the virulence of
B. pertussis
and determine if expression of the
bvg
-repressed genes is required for the virulence of
B. pertussis
, we examined the ability of
B. pertussis
mutants, defective in their ability to regulate the expression of the
bvg
-activated and/or the
bvg
-repressed genes, to cause disease in the mouse aerosol challenge model. Our results indicate that the
bvgR
-mediated regulation of gene expression contributes to respiratory infection of mice.
Entry of
Listeria monocytogenes
into Neurons Occurs by Cell-to-Cell Spread: an In Vitro Study
Authors: Dramsi S. , Lévi S. , Triller A. , Cossart P.
Published: March 2020

Listeria monocytogenes
is an intracellular pathogen that causes severe central nervous system infection in humans and animals. The ability of this bacterium to penetrate nerve cells was investigated by using rat spinal cell cultures. Entry into distinct cell types, i.e., glial cells and neurons, was monitored by a differential immunofluorescence technique with antibodies against cell type-specific markers and the bacterial pathogen.
L. monocytogenes
was detected predominantly within macrophages constituting the microglia. Astrocytes and oligodendrocytes, the major components of macroglia, were infected to a lesser extent. Surprisingly,
Listeria innocua
, a noninvasive and nonpathogenic species, also has the capacity to enter into these three types of glial cells. Entry into neurons was a very rare event. In contrast, we found that
L. monocytogenes
could efficiently invade neurons when these latter cells were cocultivated with
Listeria
-infected mouse macrophages. In this case, infection of neurons occurs by cell-to-cell spread via an
actA
-dependent mechanism. These data support the notion that infected phagocytes can be vectors by which
L. monocytogenes
gains access to privileged niches such as the central nervous system.
Role of CR4 inMycobacterium tuberculosis-Human Macrophages Binding and Signal Transduction in the Absence of Serum
Authors: Zaffran Yona , Zhang Li , Ellner Jerrold J.
Published: March 2020

The β2 integrin CR4 is involved inMycobacterium tuberculosisphagocytosis by human mononuclear phagocytes through the opsonin C3bi. In this study, we demonstrate thatM. tuberculosiscan bind directly to monocyte-derived macrophages via CR4 in the absence of any opsonins. CR4-transfected CHO cells gave similar results, suggesting recognition by CR4 of bacterial structure. Furthermore, binding ofM. tuberculosistransduced a potent signal, resulting in tyrosine phosphorylation of macrophage proteins, which was in part mediated by CR4.
Escherichia coliO157:H7 Requires Intimin for Enteropathogenicity in Calves
Authors: Dean-Nystrom Evelyn A. , Bosworth Brad T. , Moon Harley W. , O’Brien Alison D.
Published: March 2020

EnterohemorrhagicEscherichia coli(EHEC) strains require intimin to induce attaching and effacing (A/E) lesions in newborn piglets. Infection of newborn calves with intimin-positive or intimin-negative EHEC O157:H7 demonstrated that intimin is needed for colonization, A/E lesions, and disease in cattle. These results suggest that experiments to determine if intimin-based vaccines reduce O157:H7 levels in cattle are warranted.
Phase Variations of the
Mycoplasma penetrans
Main Surface Lipoprotein Increase Antigenic Diversity
Authors: Neyrolles Olivier , Chambaud Isabelle , Ferris Stéphane , Prevost Marie-Christine , Sasaki Tsuguo , Montagnier Luc , Blanchard Alain
Published: March 2020

Mycoplasma penetrans
is a recently identified mycoplasma, isolated from urine samples collected from human immunodeficiency virus (HIV)-infected patients. Its presence is significantly associated with HIV infection. The major antigen recognized during natural and experimental infections is an abundant P35 lipoprotein which, upon extraction, segregates in the Triton X-114 detergent phase and is the basis of
M. penetrans
-specific serological assays. We report here that the P35 antigen undergoes spontaneous and reversible phase variation at high frequency, leading to heterogeneous populations of mycoplasmas, even when derived from a clonal lineage. This variation was found to be determined at the transcription level, and although this property is not unique among the members of the class
Mollicutes
, the mechanism by which it occurs in
M. penetrans
differs from those previously described for other
Mycoplasma
species. Indeed, the P35 phase variation was due neither to a
p35
gene rearrangement nor to point mutations within the gene itself or its promoter. The P35 phase variation in the different variants obtained was concomitant with modifications in the pattern of other expressed lipoproteins, probably due to regulated expression of selected members of a gene family which was found to potentially encode similar lipoproteins.
M. penetrans
variants could be selected on the basis of their lack of colony immunoreactivity with a polyclonal antiserum against a Triton X-114 extract, strongly suggesting that the mechanisms involved in altering surface antigen expression might allow evasion of the humoral immune response of the infected host.
Lipopolysaccharide Enhances the Production of Vascular Endothelial Growth Factor by Human Pulp Cells in Culture
Authors: Matsushita K. , Motani R. , Sakuta T. , Nagaoka S. , Matsuyama T. , Abeyama K. , Maruyama I. , Takada H. , Torii M.
Published: March 2020

We investigated whether vascular endothelial growth factor (VEGF) production by human pulp cells (HPC) is regulated by lipopolysaccharide (LPS) in relation to the pathogenesis of pulpitis. Although HPC incubated with medium alone only marginally expressed VEGF mRNA and produced a low level of VEGF as detected by enzyme-linked immunosorbent assay, the VEGF mRNA expression and VEGF production were markedly enhanced upon stimulation with LPS fromEscherichia coli. Prevotella intermediaLPS, phorbol 12-myristate 13-acetate, and interleukin-6 also induced VEGF mRNA expression in HPC. A simian virus 40-infected HPC line also exhibited increased VEGF mRNA expression in response toE. coliLPS, but lung and skin fibroblasts did not. Fetal bovine serum (FBS) increased the sensitivity of HPC to LPS in a dose-dependent manner. HPC did not express membrane CD14 on their surfaces. However, the anti-CD14 monoclonal antibody MY4 inhibited VEGF induction upon stimulation with LPS in HPC cultures in the presence of 10% FBS but not in the absence of FBS. LPS augmented the VEGF production in HPC cultures in the presence of recombinant human soluble CD14 (sCD14). To clarify the mechanisms of VEGF induction by LPS, we examined the possible activation of the transcription factor AP-1 in HPC stimulated with LPS, by a gel mobility shift assay. AP-1 activation in HPC was clearly observed, whereas that in skin fibroblasts was not. The AP-1 inhibitor curcumin strongly inhibited LPS-induced VEGF production in HPC cultures. In addition, a protein synthesis inhibitor, cycloheximide, inhibited VEGF mRNA accumulation in response to LPS. These results suggest that the enhanced production of VEGF in HPC induced by LPS takes place via an sCD14-dependent pathway which requires new protein synthesis and is mediated in part through AP-1 activation.
Molecular Characterization of aHaemaphysalis longicornisTick Salivary Gland-Associated 29-Kilodalton Protein and Its Effect as a Vaccine against Tick Infestation in Rabbits
Authors: Mulenga Albert , Sugimoto Chihiro , Sako Yasuhito , Ohashi Kazuhiko , Musoke Anthony , Shubash Mozaria , Onuma Misao
Published: March 2020

The use of tick vaccines in mammalian hosts has been shown to be the most promising alternative tick control method to current use of acaricides, which suffers from a number of limitations. However, the success of this method is dependent on the identification, cloning, and in vitro expression of tick molecules involved in the mediation of key physiological roles with respect to the biological success of a tick as a vector and pest. We have sequenced and characterized aHaemaphysalis longicornistick salivary gland-associated cDNA coding for a 29-kDa extracellular matrix-like protein. This protein is expressed in both unfed and fed immature and matureH. longicornisticks. The predicted amino acid sequence of p29 shows high homology to sequences of some known extracellular matrix like-proteins with the structural conservation similar to all known collagen proteins. Immunization with the recombinant p29 conferred a significant protective immunity in rabbits, resulting in reduced engorgement weight for adult ticks and up to 40 and 56% mortality in larvae and nymphs that fed on the immunized rabbits. We speculate that this protein is associated with formation of tick cement, a chemical compound that enables the tick to remain attached to the host, and suggest a role for p29 as a candidate tick vaccine molecule for the control of ticks. We have discussed our findings with respect to the search of tick molecules for vaccine candidates.
Cryptococcus neoformans
Differential Gene Expression Detected In Vitro and In Vivo with Green Fluorescent Protein
Authors: del Poeta Maurizio , Toffaletti Dena L. , Rude Thomas H. , Sparks Sara D. , Heitman Joseph , Perfect John R.
Published: March 2020

Synthetic green fluorescent protein (GFP) was used as a reporter to detect differential gene expression in the pathogenic fungus
Cryptococcus neoformans
. Promoters from the
C. neoformans
actin, GAL7, or mating-type alpha pheromone (MFα1) genes were fused to GFP, and the resulting reporter genes were used to assess gene expression in serotype A
C. neoformans
. Yeast cells containing an integrated pACT::GFP construct demonstrated that the actin promoter was expressed during vegetative growth on yeast extract-peptone-dextrose medium. In contrast, yeast cells containing the inducible GAL7::GFP or MFα1::GFP reporter genes expressed significant GFP activity only during growth on galactose medium or V-8 agar, respectively. These findings demonstrated that the GAL7 and MFα1 promoters from a serotype D
C. neoformans
strain function when introduced into a serotype A strain. Because the MFα1 promoter is induced by nutrient deprivation and the
MATα
locus containing the MFα1 gene has been linked with virulence, yeast cells containing the pMFα1::GFP reporter gene were analyzed for GFP expression in the central nervous system (CNS) of immunosuppressed rabbits. In fact, significant GFP expression from the MFα1::GFP reporter gene was detected after the first week of a CNS infection. These findings suggest that there are temporal, host-specific cues that regulate gene expression during infection and that the MFα1 gene is induced during the proliferative stage of a CNS infection. In conclusion, GFP can be used as an effective and sensitive reporter to monitor specific
C. neoformans
gene expression in vitro, and GFP reporter constructs can be used as an approach to identify a novel gene(s) or to characterize known genes whose expression is regulated during infection.
Risk Factors in the Pathogenesis of Invasive Group A Streptococcal Infections: Role of Protective Humoral Immunity
Authors: Basma Hesham , Norrby-Teglund Anna , Guedez Yajaira , McGeer Allison , Low Donald E. , El-Ahmedy Omar , Schwartz Benjamin , Kotb Malak
Published: March 2020

An impressive change in the epidemiology and severity of invasive group A streptococcal infections occurred in the 1980s, and the incidence of streptococcal toxic shock syndrome cases continues to rise. The reason for the resurgence of severe invasive cases remains a mystery—has there been a change in the pathogen or in host protective immunity? To address these questions, we have studied 33 patients with invasive infection caused by genotypically indistinguishable M1T1 strains ofStreptococcus pyogeneswho had different disease outcomes. Patients were classified as having severe (n= 21) and nonsevere (n= 12) invasive infections based on the presence or absence of shock and organ failure. Levels of anti-M1 bactericidal antibodies and of anti-streptococcal superantigen neutralizing antibodies in plasma were significantly lower in both groups than in age- and geographically matched healthy controls (P< 0.01). Importantly, the levels of these protective antibodies in plasma samples from severe and nonsevere invasive cases were not different. Together the data suggest that low levels of protective antibodies may contribute to host susceptibility to invasive streptococcal infection but do not modulate disease outcome. Other immunogenetic factors that regulate superantigen responses may influence the severity of systemic manifestations associated with invasive streptococcal infection.
Role of Gamma Interferon in Cellular Immune Response against MurineEncephalitozoon cuniculiInfection
Authors: Khan Imtiaz A. , Moretto Magali
Published: March 2020

Microsporidia are obligate intracellular protozoan parasites that cause a wide variety of opportunistic infection in patients with AIDS. Because it is able to grow in vitro,Encephalitozoon cuniculiis currently the best-studied microsporidian. T cells mediate protective immunity against this parasite. Splenocytes obtained from infected mice proliferate in vitro in response to irradiated parasites. A transient state of hyporesponsiveness to parasite antigen and mitogen was observed at day 17 postinfection. This downregulatory response could be partially reversed by addition of nitric oxide (NO) antagonist to the culture. Mice infected withE. cuniculisecrete significant levels of gamma interferon (IFN-γ). Treatment with antibody to IFN-γ or interleukin-2 (IL-12) was able to neutralize the resistance to the parasite. Mutant animals lacking the IFN-γ or IL-12 gene were highly susceptible to infection. However, mice unable to secrete NO withstood high doses of parasite challenge, similar to normal wild-type animals. These studies describe an IFN-γ-mediated protection againstE. cuniculiinfection that is independent of NO production.
Effect of Temperature on Growth, Hemagglutination, and Protease Activity ofPorphyromonas gingivalis
Authors: Percival Rimondia S. , Marsh Philip D. , Devine Deirdre A. , Rangarajan Minnie , Aduse-Opoku Joseph , Shepherd Philip , Curtis Michael A.
Published: March 2020

Bacteria persisting in periodontal pockets are exposed to elevated temperatures during periods of inflammation. Temperature is an environmental factor that can modulate gene expression. Consequently, in the present study we examined the effect of temperature on the expression of virulence determinants by the periodontopathogen,Porphyromonas gingivalis. P. gingivalisW50 was grown in a complex medium under hemin excess at pH 7.0 and at a constant temperature of either 37, 39, or 41°C; cultures were monitored for protease and hemagglutinin activity.P. gingivalisgrew well at all three temperatures. An increase in growth temperature from 37 to 39°C resulted in a 65% reduction in both total arginine- and lysine-specific activities (P< 0.01). A further rise in growth temperature to 41°C led to even greater reductions in arginine-specific (82%;P< 0.001) and lysine-specific (73%;P< 0.01) activities. These reductions were also associated with an altered distribution of individual arginine-specific enzyme isoforms. At 41°C, there was a disproportionate reduction in the level of the heterodimeric RI protease, which also contains adhesin domains. The reduction also correlated with a markedly diminished hemagglutination activity of cells, especially in those grown at 41°C, and a reduced immunoreactivity with a monoclonal antibody which recognizes gene products involved in hemagglutination. Thus, as the environmental temperature increased,P. gingivalisadopted a less aggressive phenotype, while retaining cell population levels. The coordinate down-regulation of virulence gene expression in response to an environmental cue linked to the intensity of the host inflammatory response is consistent with the clinically observed cyclical nature of disease progression in periodontal diseases.
Adaptive Immune Response toShigella flexneri2acydCin Immunocompetent Mice and Mice Lacking Immunoglobulin A
Authors: Way Sing Sing , Borczuk Alain C. , Goldberg Marcia B.
Published: March 2020

Shigella flexneri cydC, which is deficient in cytochromebd, was rapidly cleared from the lungs of intranasally inoculated mice and was Sereny negative, yet it induced 93% protection against challenge with wild-typeS. flexneri. Mice that lack immunoglobulin A (IgA) were fully protected, suggesting that IgA may not be required for adaptive immunity in this model system.
Biological Properties of Structurally Related α-Helical Cationic Antimicrobial Peptides
Authors: Scott Monisha G. , Yan Hong , Hancock Robert E. W.
Published: March 2020

A series of α-helical cationic antimicrobial peptide variants with small amino acid changes was designed. Alterations in the charge, hydrophobicity, or length of the variant peptides did not improve the antimicrobial activity, and there was no statistically significant correlation between any of these factors and the MIC for
Pseudomonas aeruginosa
,
Escherichia coli
, or
Salmonella typhimurium
. Individual peptides demonstrated synergy with conventional antibiotics against antibiotic-resistant strains of
P. aeruginosa
. The peptides varied considerably in the ability to bind
E. coli
O111:B4 lipopolysaccharide (LPS), and this correlated significantly with their antimicrobial activity and ability to block LPS-stimulated tumor necrosis factor and interleukin-6 production. In general, the peptides studied here demonstrated a broad range of activities, including antimicrobial, antiendotoxin, and enhancer activities.
Biological Effects of
Pseudomonas aeruginosa
Type III-Secreted Proteins on CHO Cells
Authors: Vallis Amy J. , Finck-Barbançon Viviane , Yahr Timothy L. , Frank Dara W.
Published: March 2020

A strain of
Pseudomonas aeruginosa
that fails to express known type III-secreted effector proteins was constructed as an expression host. Individual effectors were expressed in
trans
, and their biological effects on CHO cells were assessed in an acute cellular infection model. Intoxication with ExoS, ExoT, or ExoY resulted in alterations in cell morphology. As shown in previous genetic studies, ExoU expression was linked to acute cytotoxicity.
uspB
, a New ς
S
-Regulated Gene in
Escherichia coli
Which Is Required for Stationary-Phase Resistance to Ethanol
Authors: Farewell Anne , Kvint Kristian , Nyström Thomas
Published: March 2020

The open reading frame immediately upstream of
uspA
is demonstrated to encode a 14-kDa protein which we named UspB (universal stress protein B) because of its general responsiveness to different starvation and stress conditions. UspB is predicted to be an integral membrane protein with at least one and perhaps two membrane-spanning domains. Overexpression of UspB causes cell death in stationary phase, whereas mutants of
uspB
are sensitive to exposure to ethanol but not heat in stationary phase. In contrast to
uspA
, stationary-phase induction of
uspB
requires the sigma factor ς
S
. The expression of
uspB
is modulated by H-NS, consistent with the role of H-NS in altering ς
S
levels. Our results demonstrate that a gene of the RpoS regulon is involved in the development of stationary-phase resistance to ethanol, in addition to the regulon’s previously known role in thermotolerance, osmotolerance, and oxidative stress resistance.
The General Stress Sigma Factor ς
S
of
Escherichia coli
Is Induced during Diauxic Shift from Glucose to Lactose
Authors: Fischer Daniela , Teich Antje , Neubauer Peter , Hengge-Aronis Regine
Published: March 2020

The general stress sigma factor ς
S
(RpoS) of
Escherichia coli
is strongly induced in response to glucose starvation. This increase in the cellular ς
S
level is due to stabilization of ς
S
, which under non-stress conditions is subject to rapid proteolysis. In the present study, it is demonstrated that ς
S
is also induced during the diauxic shift from glucose to lactose, i.e., under conditions of glucose exhaustion in the presence of another, less-preferred carbon source that eventually gets utilized. This ς
S
induction, which is due to stabilization, is transient and precedes the induction of β-galactosidase. In parallel, ς
S
-dependent genes are transiently activated, as was shown here for
osmY
. Although ς
S
can mediate transcription of
lacZ
in vitro, ς
S
does not contribute to the induction of β-galactosidase during the diauxic lag phase. Rather, the induction of ς
S
and the general stress response during the diauxic shift plays the role of a rapidly activated emergency system, which is shut off again as soon as the cells are able to cope with the stress situation by utilizing a more specific and more economical system.
Role and Regulation of
Bacillus subtilis
Glutamate Dehydrogenase Genes
Authors: Belitsky Boris R. , Sonenshein Abraham L.
Published: March 2020

The complete
Bacillus subtilis
genome contains two genes with the potential to encode glutamate dehydrogenase (GlutDH) enzymes. Mutations in these genes were constructed and characterized. The
rocG
gene proved to encode a major GlutDH whose synthesis was induced in media containing arginine or ornithine or, to a lesser degree, proline and was repressed by glucose. A
rocG
null mutant was impaired in utilization of arginine, ornithine, and proline as nitrogen or carbon sources. The
gudB
gene was expressed under all growth conditions tested but codes for a GlutDH that seemed to be intrinsically inactive. Spontaneous mutations in
gudB
that removed a 9-bp direct repeat within the wild-type
gudB
sequence activated the GudB protein and allowed more-efficient utilization of amino acids of the glutamate family.
A Homolog of CcpA Mediates Catabolite Control in
Listeria monocytogenes
but Not Carbon Source Regulation of Virulence Genes
Authors: Behari Jaideep , Youngman Philip
Published: March 2020

Readily utilizable sugars down-regulate virulence gene expression in
Listeria monocytogenes
, which has led to the proposal that this regulation may be an aspect of global catabolite regulation (CR). We recently demonstrated that the metabolic enzyme α-glucosidase is under CR in
L. monocytogenes
. Here, we report the cloning and characterization from
L. monocytogenes
of an apparent ortholog of
ccpA
, which encodes an important mediator of CR in several low-G+C-content gram-positive bacteria.
L. monocytogenes ccpA
(
ccpA
Lm
) is predicted to encode a 335-amino-acid protein with nearly 65% identity to the gene product of
Bacillus subtilis ccpA
(
ccpA
Bs
). Southern blot analysis with a probe derived from
ccpA
Lm
revealed a single strongly hybridizing band and also a second band of much lower intensity, suggesting that there may be other closely related sequences in the
L. monocytogenes
chromosome, as is the case in
B. subtilis
. Disruption of
ccpA
Lm
resulted in the inability of the mutant to grow on glucose-containing minimal medium or increase its growth rate in the presence of preferred sugars, and it completely eliminated CR of α-glucosidase activity in liquid medium. However, α-glucosidase activity was only partially relieved from CR on solid medium. These results suggest that
ccpA
is an important element of carbon source regulation in
L. monocytogenes
. Nevertheless, utilizable sugars still down-regulate the expression of
hly
, which encodes the virulence factor hemolysin, in a
ccpA
Lm
mutant, indicating that CcpA is not involved in carbon source regulation of virulence genes.
N -Acyl-
l
-Homoserine Lactone Autoinducers Control Production of an Extracellular Lipopeptide Biosurfactant Required for Swarming Motility of
Serratia liquefaciens
MG1
Authors: Lindum P. W. , Anthoni U. , Christophersen C. , Eberl L. , Molin S. , Givskov M.
Published: March 2020

A nonswarming
Serratia liquefaciens
mutant deficient in serrawettin W2 production was constructed by transposon mutagenesis. Sequence homology indicated that insertion had occurred in gene
swrA
, which encodes a putative peptide synthetase. Expression of
swrA
is controlled by quorum sensing.
Monoclonal Antibodies Directed against Conserved Epitopes on the Nucleocapsid Protein and the Major Envelope Glycoprotein of Equine Arteritis Virus
Authors: Weiland Emilie , Bolz Sylvia , Weiland Frank , Herbst Werner , Raamsman Martin J. B. , Rottier Peter J. M. , De Vries Antoine A. F.
Published: March 2020

We recently developed a highly effective immunization procedure for the generation of monoclonal antibodies (MAbs) directed against the porcine reproductive and respiratory syndrome virus (E. Weiland, M. Wieczorek-Krohmer, D. Kohl, K. K. Conzelmann, and F. Weiland, Vet. Microbiol. 66:171–186, 1999). The same method was used to produce a panel of 16 MAbs specific for the equine arteritis virus (EAV). Ten MAbs were directed against the EAV nucleocapsid (N) protein, and five MAbs recognized the major viral envelope glycoprotein (G
L
). Two of the EAV G
L
-specific MAbs and one antibody of unknown specificity neutralized virus infectivity. A comparison of the reactivities of the MAbs with 1 U.S. and 22 newly obtained European field isolates of EAV demonstrated that all N-specific MAbs, the three nonneutralizing anti-G
L
MAbs, and the weakest neutralizing MAb (MAb E7/d15-c9) recognized conserved epitopes. In contrast, the two MAbs with the highest neutralization titers bound to 17 of 23 (MAb E6/A3) and 10 of 23 (MAb E7/d15-c1) of the field isolates. Ten of the virus isolates reacted with only one of these two MAbs, indicating that they recognized different epitopes. The G
L
-specific MAbs and the strongly neutralizing MAb of unknown specificity (MAb E6/A3) were used for the selection of neutralization-resistant (NR) virus variants. The observation that the E6/A3-specific NR virus variants were neutralized by MAb E7/d15-c1 and that MAb E6/A3 blocked the infectivity of the E7/d15-c1-specific NR escape mutant confirmed that these antibodies reacted with distinct antigenic sites. Immunoelectron microscopy revealed for the first time that the antigenic determinants recognized by the anti-G
L
MAbs were localized on the virion surface. Surprisingly, although the immunofluorescence signal obtained with the neutralizing antibodies was relatively weak, they mediated binding of about three times as much gold granules to the viral envelope than the nonneutralizing anti-G
L
MAbs.
Use of PCR with Universal Primers and Restriction Endonuclease Digestions for Detection and Identification of Common Bacterial Pathogens in Cerebrospinal Fluid
Authors: Lu Jang-Jih , Perng Cherng-Lih , Lee Shih-Yi , Wan Chih-Chieng
Published: March 2020

We have designed a universal PCR capable of amplifying a portion of the 16S rRNA gene of eubacteria, including
Staphylococcus aureus
,
Staphylococcus epidermidis
,
Streptococcus pyogenes
,
Streptococcus agalactiae
,
Streptococcus pneumoniae
,
Enterococcus faecium
,
Enterococcus faecalis
,
Mycobacterium tuberculosis
,
Legionella pneumophila
,
Escherichia coli
,
Klebsiella pneumoniae
,
Serratia marcescens
,
Enterobacter cloacae
,
Pseudomonas aeruginosa
,
Acinetobacter baumannii
,
Proteus mirabilis
,
Haemophilus influenzae
, and
Neisseria meningitidis
. The sizes of the amplified products from various bacteria were the same (996 bp), but the restriction patterns of most PCR products generated by
Hae
III digestion were different. PCR products from
S. aureus
and
S. epidermidis
could not be digested by
Hae
III but yielded different patterns when they were digested with
Mnl
I. PCR products from
S. pneumoniae
,
E. faecium
, and
E. faecalis
yielded the same
Hae
III digestion pattern but could be differentiated by
Alu
I digestion. PCR products from
E. coli
,
K. pneumoniae
,
S. marcescens
, and
E. cloacae
also had the same
Hae
III digestion pattern but had different patterns when digested with
Dde
I or
Bst
BI. This universal PCR could detect as few as 10
E. coli
or 250
S. aureus
organisms. Compared with culture, the sensitivity of this universal PCR for detection and identification of bacteria directly from 150 cerebrospinal fluids was 92.3%. These results suggest that this universal PCR coupled with restriction enzyme analysis can be used to detect and identify bacterial pathogens in clinical specimens.
False Molecular Clusters due to Nonrandom Association of IS
6110
with
Mycobacterium tuberculosis
Authors: Gillespie S. H. , Dickens A. , McHugh T. D.
Published: March 2020

We sought to determine whether nonrandom association of IS
6110
with
Mycobacterium tuberculosis
could result in false-positive clustering in unselected collections of isolates. We typed 196 strains of
M. tuberculosis
from an unselected community-based study in northern Tanzania by IS
6110
and polymorphic GC-rich repetitive-sequence (PGRS) methodologies. The strains were analyzed by Gelcompar computer software. Analysis of 13 out of 25 groups showed that isolates with identical IS
6110
and PGRS patterns were likely to be the same strain. Some IS
6110
groups containing strains with identical PGRS patterns had similar IS
6110
patterns that differed only by movement of the element. Isolates assigned to a single group (i.e., group 11) on the basis of sharing an identical IS
6110
fingerprint pattern did not share identical PGRS fingerprint patterns. Six out of the nine bands in these isolates were in hot-spot locations, as previously defined. This indicates that nonrandom association may result in false-positive clustering in unselected community-based studies. Only strains with identical PGRS and IS
6110
patterns are likely to be recently transmitted.
High Rate of Tetracycline Resistance in
Streptococcus pyogenes
in Iran: an Epidemiological Study
Authors: Jasir A. , Tanna A. , Noorani A. , Mirsalehian A. , Efstratiou A. , Schalen C.
Published: March 2020

Streptococcus pyogenes
, a major human pathogen, is still considered susceptible to beta-lactams, but for other relevant antibiotics, highly variable resistance rates have been reported. Since no data were available from Iran, we tested 1,335 throat isolates from two different regions of the country for their antibiotic susceptibilities and, for comparison, a collection of 80 strains isolated from 1989 to 1991. Erythromycin resistance was uncommon (0.6%), whereas an overall high rate of tetracycline resistance was found, increasing between 1989–1991 and 1995–1997 from 23 to 42%. The tetracycline-resistant strains belonged to more than 10 different T types, the majority being types 4, 11, and B3264. By conventional M typing of 406 tetracycline-resistant isolates, more than 20 different M types were found. Approximately 50% of the strains were nontypeable by T agglutination as well as serological M typing; however, by genotyping by a combined PCR–capture-enzyme-linked immunosorbent assay, many of these strains were successfully
emm
typed. We conclude that the high rate of tetracycline resistance among Iranian
S. pyogenes
isolates is due to multiclonal dissemination of resistance within the streptococcal population rather than epidemic spread of single clones.
Isolation and Characterization of Glycopeptide-Resistant Enterococci from Hospitalized Patients over a 30-Month Period
Authors: Nelson R. R. S. , McGregor K. F. , Brown A. R. , Amyes S. G. B. , Young H.-K.
Published: March 2020

In February 1996, a Hospital Infection Control Practices Advisory Committee-style screening program was commenced to isolate and subsequently characterize glycopeptide-resistant enterococci (GRE) from patients at a hospital trust in Glasgow, Scotland. Over the next 30 months, GRE were isolated from 154 patients. GRE were isolated from patients in traditionally high-risk areas such as the renal unit and intensive care unit and also in areas considered to be lower risk, including medical wards and associated long-stay geriatric hospitals. The majority (90%) of isolates were
Enterococcus faecium vanB
. The remaining isolates consisted of seven
E. faecalis
(
vanA
), three
E. gallinarum
(
vanC
), and a further six
E. faecium
(five
vanA
, one both
vanA
and
vanB
) isolates. Analysis of
Sma
I-digested DNA by pulsed-field gel electrophoresis revealed that 34 of 40 (85%) VanB
E. faecium
isolates were identical or closely related, while 11 of 13 (85%) VanA GRE were distinct. High-level aminoglycoside resistance was seen in less than 8% of isolates. VanB
E. faecium
isolates were almost uniformly resistant to ampicillin and tetracycline. In this study, GRE have been isolated over a prolonged period from a broad range of patients. Glycopeptide resistance within the study hospital trust appeared to be mainly due to the clonal dissemination of a single strain of
E. faecium
VanB.
Molecular Characteristics and Epidemiological Significance of Shiga Toxin-Producing
Escherichia coli
O26 Strains
Authors: Zhang Wen-Lan , Bielaszewska Martina , Liesegang Almut , Tschäpe Helmut , Schmidt Herbert , Bitzan Martin , Karch Helge
Published: March 2020

Fifty-five Shiga toxin (Stx)-producing
Escherichia coli
(STEC) O26:H11 and O26:H
−
strains isolated from humans between 1965 and 1999 in Germany and the Czech Republic were investigated for their chromosomal and plasmid characteristics. All motile (
n
= 23) and nonmotile (
n
= 32) STEC O26 strains were shown to possess the identical flagellin subunit-encoding gene (
fliC
). We observed a striking recent shift of the
stx
genotype from

stx
1

to

stx
2

among the STEC O26 isolates. While

stx
1

was the exclusive genotype identified in our collection until 1994, 94% of the isolates obtained after 1997 possessed

stx
2

either alone (71%) or together with

stx
1

(23%). Plasmid profiling demonstrated a remarkable heterogeneity with respect to plasmid sizes and combinations. Southern blot analysis of plasmid DNA with probes specific to potential accessory virulence genes revealed considerable additional variability in gene composition and arrangement. Pulsed-field gel electrophoresis (PFGE) differentiated 16 subgroups among the 55 STEC O26 strains. Using these techniques we demonstrate the emergence of a new clonal subgroup characterized by PFGE pattern A and a unique combination of virulence markers including

stx
2

and a single, approximately 90-kb plasmid harboring the enterhemorrhagic
E. coli hlyA
and
etp
genes. The proportion of PFGE subgroup A strains among STEC O26 isolates rose from 30% in 1996 to more than 50% in 1999. Four clusters of infections with the clonal subgroup A were identified. We conclude that the STEC serogroup O26 is diverse and that pathogenic clonal subgroups can rapidly emerge during short intervals. The extensive genetic diversity of STEC O26 provides a basis for molecular subtyping of this important non-O157 STEC serogroup.
PCR-Based Quantification ofBorrelia burgdorferiOrganisms in Canine Tissues over a 500-Day Postinfection Period
Authors: Straubinger Reinhard K.
Published: March 2020

Borrelia burgdorferiinfection in beagle dogs was studied quantitatively with skin punch biopsy samples and blood samples collected at 4- and 2-week intervals, respectively, over a 500-day period. Thereafter, 25 tissue samples of each dog were collected for further analysis. Starting at day 120 after tick challenge, 12 dogs were treated with antibiotics (azithromycin, ceftriaxone, or doxycycline) for 30 consecutive days. Four dogs received no antibiotic therapy. Quantification ofB. burgdorferiDNA was done with an ABI Prism 7700 Sequence Detection System with oligonucleotide primers and a fluorescence-labeled probe designed to specifically amplify a fragment of theospAgene ofB. burgdorferistrain N40. All 16 dogs became infected withB. burgdorferiafter tick challenge. In skin biopsy samples, spirochete numbers peaked at day 60 postinfection (<1.5 × 106organisms per 100 μg of extracted DNA), at the same time when clinical signs of arthritis developed in 11 of 16 dogs, and decreased to almost undetectable levels during the following 6 months. The number ofB. burgdorferiorganisms detected in skin biopsy samples was inversely correlated with the antibody levels measured by enzyme-linked immunosorbent assay. Antibiotic treatment reduced the amount of detectable spirochete DNA in skin tissue by a factor of 1,000 or more. At the end of the experiment,B. burgdorferiDNA was detectable at low levels (102to 104organisms per 100 μg of extracted DNA) in multiple tissue samples regardless of treatment. However, more tissue samples of untreated dogs than of antibiotic-treated dogs were positive, and tissue samples of untreated dogs also were positive by culture. Only 1.6% of 576 blood samples of all dogs were positive forB. burgdorferiby PCR.
Misidentifying Helicobacters: the
Helicobacter cinaedi
Example
Authors: Vandamme Peter , Harrington Clare S. , Jalava Katri , On Stephen L. W.
Published: March 2020

Whole-cell protein electrophoresis and biochemical examination by means of a panel of 64 tests were used to identify 14 putative helicobacters to the species level. The results were confirmed by means of DNA-DNA hybridization experiments and were used to discuss misidentification of helicobacters based on 16S rRNA gene sequence data. The data indicated that comparison of near-complete 16S ribosomal DNA sequences does not always provide conclusive evidence for species level identification and may prove highly misleading. The data also indicated that “
Helicobacter westmeadii
” is a junior synonym of
Helicobacter cinaedi
and that
Helicobacter
sp. strain Mainz belongs to the same species.
H. cinaedi
occurs in various animal reservoirs, including hamsters, dogs, cats, rats, and foxes. Appropriate growth conditions and identification strategies will be required to establish the genuine significance of this widely distributed
Helicobacter
species.
Comparative Evaluation of Three Different Genotyping Methods for Investigation of Nosocomial Outbreaks of Legionnaires' Disease in Hospitals
Authors: Jonas Daniel , Meyer Heinz-Georg W. , Matthes Philipp , Hartung Doris , Jahn Bernhard , Daschner Franz D. , Jansen Bernd
Published: March 2020

The increased incidence of nosocomial Legionnaires' disease in two hospitals prompted investigation of possible environmental sources. In the search for an effective DNA-typing technique for use in hospital epidemiology, the performance and convenience of three methods—
Sfi
I macrorestriction analysis (MRA), amplified fragment length polymorphism (AFLP), and arbitrarily primed PCR (AP-PCR)—were compared. Twenty-nine outbreak-associated and eight nonassociated strains of
Legionella pneumophila
with 13 MRA types and subtypes were investigated. These strains comprised isolates from bronchoalveolar lavages, from environmental, patient-related sources, and type strains. All three typing methods detected one predominant genotype associated with the outbreaks in both hospitals. All of them correctly assigned epidemiologically associated, environmental isolates to their respective patient specimens. AP-PCR was the least discriminating and least reproducible technique. In contrast, AFLP was demonstrated as being the method with the best interassay reproducibility (90%) and concordance (94%) in comparison to the genotyping standard of MRA and the epidemiological data. Analysis of AFLP fragments revealed 12 different types and subtypes. Because of its simplicity and reproducibility, AFLP proved to be the most effective technique in outbreak investigation.