bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of an Insertion Sequence Located in a Region Encoding Virulence Factors of
Streptococcus pyogenes
Authors: Berge Andreas , Rasmussen Magnus , Björck Lars
Published: December 2019

An insertion sequence, IS
1562
, was identified in a
Streptococcus pyogenes
strain of the clinically important M1 serotype. IS
1562
is located in the
mga
regulon between the genes coding for the M protein and the C5a peptidase, both important virulence factors. The same or similar insertion sequences were found in most
S. pyogenes
strains, but the chromosomal location differed among isolates.
Inhibition of Class II Major Histocompatibility Complex Antigen Processing byEscherichia coliHeat-Labile Enterotoxin Requires an Enzymatically Active A Subunit
Authors: Matousek Milita P. , Nedrud John G. , Cieplak Witold , Harding Clifford V.
Published: December 2019

Escherichia coliheat-labile enterotoxin (LT) and cholera toxin (CT) were found to inhibit intracellular antigen processing. Processing was not inhibited by mutant LT with attenuated ADP-ribosyltransferase activity, CT B or LT B subunit, which enhanced presentation of preexisting cell surface peptide-class II major histocompatibility complex complexes. Inhibition of antigen processing correlated with A subunit ADP-ribosyltransferase activity.
Altered Cytokine Production and Impaired Antimycobacterial Immunity in Protein-Malnourished Guinea Pigs
Authors: Dai Guixiang , McMurray David N.
Published: December 2019

Protein malnutrition leads to multiple detrimental alterations of host immune responses to mycobacterial infection. In this study, we demonstrated that splenocytes from low-protein (LP) guinea pigs vaccinated 6 weeks previously with attenuatedMycobacterium tuberculosisH37Ra failed to control the accumulation of virulentM. tuberculosisH37Rv in cocultured autologous peritoneal macrophages, despite the fact that they were able to control the accumulation of virulent tubercle bacilli in cocultured syngeneic peritoneal macrophages from normally nourished guinea pigs as successfully as did those from high-protein (HP) counterparts. Vaccine-induced growth control of virulentM. tuberculosisH37Rv in these cocultures appeared to be mediated by CD4 lymphocytes but not CD8 cells. Tuberculin (purified protein derivative [PPD])-induced lymphoproliferation was markedly impaired in vaccinated LP guinea pigs, and the depletion of CD4 lymphocytes significantly decreased lymphocyte proliferation whereas CD8 cell depletion did not. Protein malnutrition also impaired the abilities of cells from vaccinated LP guinea pigs to produce cytokines, including interferon, tumor necrosis factor alpha (TNF-α) and transforming growth factor beta (TGF-β), in response to PPD, despite the demonstration of higher serum levels of TNF-α and TGF-β after an intravenous injection of PPD into LP guinea pigs. In contrast, peritoneal macrophages from protein-malnourished guinea pigs produced a higher level of TGF-β 4 days after infection in vitro withM. tuberculosisH37Rv than did those from protein adequate controls. These results suggest that dietary protein malnutrition impairs vaccine-induced resistance toM. tuberculosis, in part, by altering the cytokine profile to favor macrophage deactivation.
Molecular Basis for Antigenic Variation of a Protective Strain-Specific Antigen of
Ehrlichia risticii
Authors: Biswas Biswajit , Vemulapalli Ramesh , Dutta Sukanta K.
Published: December 2019

Ehrlichia risticii
, the causative agent of Potomac horse fever, has recently been isolated from many vaccinated horses with typical clinical signs of the disease. The heterogeneity of the
E. risticii
isolates obtained from the vaccinated horses necessitates the identification of the molecular basis of strain variations to elucidate the vaccine failure and to aid in the development of an efficient vaccine against this disease. As an attempt, two major cross-reacting surface antigen genes of 50- and 85-kDa antigens, present separately in strains 25-D (isolated in 1984) and 90-12 (isolated in 1990 from a vaccinated horse), respectively, were cloned and sequenced. A comparative sequence analysis revealed differences and similarities between these two antigens with strain-specific sizes (SSA). The 2.5- and 1.6-kb genes coding for the 85- and 50-kDa proteins, respectively, contained many different tandem repeats. The identical repeat motifs were more frequent in the middle of both genes, but the numbers and positions of the repeats were altogether different in the genes. Many of these direct repeats of both genes had exact sequence homology and coded for the same amino acids. The homology of the 5′- and 3′-flanking regions of the two genes was greater than that of the regions in the central part of the genes. A comparative analysis of the deduced amino acid sequences of these two antigen genes indicated eight common domains, which were designated identical domains. Although the sequence homologies of these identical domains were the same, the positions of the domains in their respective strains were completely different. This finding might be one of the bases of antigenic variation between the strains. In addition, there were a few unique regions in both antigen genes where no sequence homology existed. These specific regions were designated unique domains. The 50-kDa protein had two such unique domains, and the 85-kDa protein had six such unique domains. The presence of such unique domains contributed to the large size variation of these SSA. The cross-reactivity of recombinant proteins confirmed the presence of conserved epitopes between these two antigens. The SSA have been determined to be apparent protective antigens of
E. risticii
.
Phospholipid Composition of PurifiedChlamydia trachomatisMimics That of the Eucaryotic Host Cell
Authors: Hatch Grant M. , McClarty Grant
Published: December 2019

Chlamydia trachomatisis an obligate intracellular eubacterial parasite capable of infecting a wide range of eucaryotic host cells. Purified chlamydiae contain several lipids typically found in eucaryotes, and it has been established that eucaryotic lipids are transported from the host cell to the parasite. In this report, we examine the phospholipid composition ofC. trachomatispurified from host cells grown under a variety of conditions in which the cellular phospholipid composition was altered. A mutant CHO cell line, with a thermolabile CDP-choline synthetase, was used to show that decreased host cell phosphatidylcholine levels had no significant effect onC. trachomatisgrowth. However, less phosphatidylcholine was transported to the parasite and purified elementary bodies contained decreased levels of phosphatidylcholine. Brefeldin A, fumonisin B1, and exogenous sphingomyelinase were used to alter levels of host cell sphingomyelin. None of the agents had a significant effect onC. trachomatisreplication. Treatment with fumonisin B1and exogenous sphingomyelinase resulted in decreased levels of host cell sphingomyelin. This had no effect on glycerophospholipid trafficking to chlamydiae; however, sphingomyelin trafficking was reduced and elementary bodies purified from treated cells had reduced sphingomyelin content. Exposure to brefeldin A, which had no adverse effect on chlamydia growth, resulted in an increase in cellular levels of sphingomyelin and a concomitant increase in the amount of sphingomyelin in purified chlamydiae. Under the experimental conditions used, brefeldin A treatment had only a small effect on sphingomyelin trafficking to the host cell surface or toC. trachomatis. Thus, the final phospholipid composition of purifiedC. trachomatismimics that of the host cell in which it is grown.
Induction of the Lysogenic Phage Encoding Cholera Toxin in Naturally Occurring Strains of Toxigenic
Vibrio cholerae
O1 and O139
Authors: Faruque Shah M. , Asadulghani  , Abdul Alim A. R. M. , Albert M. John , Nasirul Islam K. M. , Mekalanos John J.
Published: December 2019

In toxigenic
Vibrio cholerae
, the CTX genetic element which carries the genes for cholera toxin (CT) is the genome of a lysogenic bacteriophage (CTXΦ). Clinical and environmental strains of
V. cholerae
O1 or O139 and stools that were culture positive for cholera were analyzed to study the induction and transmission of CTXΦ. To our knowledge, this is the first report of the examination of CTXΦ in clinical materials and in naturally occurring strains. DNA probe analysis revealed that 4.25% (6 of 141) of the isolated
V. cholerae
strains spontaneously produced a detectable level of extracellular CTXΦ particles in the culture supernatants whereas another 34.04% (48 of 141) produced CTXΦ particles when induced with mitomycin C. CTXΦ isolated from 10 clinical or environmental strains infected a CT-negative recipient strain, CVD103, both inside the intestines of infant mice and under laboratory conditions. All culture-positive stools analyzed were negative for the presence of CTXΦ both in the DNA probe assay and by in vivo assay for the infection of the recipient strain in infant mice. These results suggested that naturally occurring strains of toxigenic
V. cholerae
are inducible lysogens of CTXΦ but that cholera pathogenesis in humans is not associated with the excretion of CTXΦ particles in stools, indicating that induction of the phage may not occur efficiently inside the human intestine. However, in view of the efficient transmission of the phage under conditions conducive to the expression of toxin-coregulated pili, it appears that propagation of CTXΦ in the natural habitat may involve both environmental and host factors.
Interactions of the Invasive PathogensSalmonella typhimurium,Listeria monocytogenes, andShigella flexneriwith M Cells and Murine Peyer’s Patches
Authors: Jensen V. Behrana , Harty John T. , Jones Bradley D.
Published: December 2019

Invasive enteric bacteria must pass through the intestinal epithelium in order to establish infection. It is becoming clear that a common target for intestinal mucosa penetration is the specialized epithelial cell of Peyer’s patches, the M cell. In order to gain a better understanding of how bacteria interact with M cells, we have compared the interactions ofSalmonella typhimurium,Listeria monocytogenes, andShigella flexneriwith M cells by using a murine ligated-loop model. Our results indicate thatS. typhimuriumpossesses a highly efficient mechanism for M cell entry that targets and destroys these cells, whileL. monocytogenesandS. flexneriappear to be internalized into M cells in a less disruptive fashion. Early uptake ofListeriaorShigellainto M cells appeared to lead to the death of some cells, as evidenced by the appearance of holes in the intestinal epithelium. At later time points, the follicle-associated epithelium of animals infected with these bacteria displayed extensive destruction. These data indicate that enteric pathogens use different strategies to interact with M cells and initiate infection of a host.
Magnesium and the Role of
mgtC
in Growth of
Salmonella typhimurium
Authors: Moncrief Mary Beth C. , Maguire Michael E.
Published: December 2019

Salmonella typhimurium
has three distinct transport systems for Mg
2+
: CorA, MgtA, and MgtB. The
mgtCB
operon encodes two proteins, MgtC, a hydrophobic protein with a predicted molecular mass of 22.5 kDa, and MgtB, a 102-kDa P-type ATPase Mg
2+
transport protein. The
mgtCB
locus has been identified as part of a new
Salmonella
pathogenicity island, SPI-3. Transcription of
mgtCB
is regulated by extracellular Mg
2+
via the two-component PhoPQ regulatory system important for virulence. To elucidate MgtC’s role in a low-Mg
2+
environment, we looked at growth and transport in strains lacking the CorA and MgtA Mg
2+
transporters but expressing MgtB, MgtC, or both.

mgtC mgtB
+

and

mgtC
+
mgtB
+

strains exhibited growth in N minimal medium without added Mg
2+
with a 1- to 2-h lag phase. An

mgtC
+
mgtB

strain was also able to grow in N minimal medium without added Mg
2+
but only after a 24-h lag phase. In N minimal medium containing 10 mM Mg
2+
, all strains grew after a short lag phase; the

mgtC
+
mgtB

strain grew to a higher optical density at 600 nm than an

mgtC
+
mgtB
+

strain and was comparable to wild type. The lengthy lag phase before growth in an

mgtC
+
mgtB

strain was not due to lack of expression of MgtC. Western blot analysis indicated that substantial MgtC protein is present by 2 h after suspension in N minimal medium. Surprisingly, in an

mgtC
+
mgtB
+

strain, MgtC was undetectable during Mg
2+
starvation, although large amounts of MgtB were observed. The lack of expression of MgtC is not dependent on functional MgtB, since a strain carrying a nonfunctional MgtB with a mutation (D379A) also did not make MgtC. Since, during invasion of eukaryotic cells,
S. typhimurium
appears to be exposed to a low-pH as well as a low-Mg
2+
environment, the growth of an

mgtC
+
mgtB

strain was tested at low pH with and without added Mg
2+
. While significant quantities of MgtC could be detected after suspension at pH 5.2, the

mgtC
+
mgtB

strain was unable to grow at pH 5.2 whether or not Mg
2+
was present. Finally, using
63
Ni
2+
and
57
Co
2+
as alternative substrates for the unavailable
28
Mg
2+
, cation uptake could not be detected in an

mgtC
+
mgtB

strain after Mg
2+
starvation. We conclude that MgtC is not a Mg
2+
transporter and that it does not have a primary role in the survival of
S. typhimurium
at low pH.
Identification and Characterization of a Phase-Specific, Nuclear DNA Binding Protein from the Dimorphic Pathogenic Fungus
Histoplasma capsulatum
Authors: Abidi Fatima E. , Roh Haeri , Keath Elizabeth J.
Published: December 2019

Genes expressed in the parasitic yeast (Y) phase of the dimorphic fungal pathogen
Histoplasma capsulatum
which are transcriptionally silent in the mycelial (M) phase have recently been cloned and analyzed. To understand the molecular regulation of genes involved in the transition to and maintenance of the Y phase, the presumptive 5′ regulatory regions of two Y phase-specific genes (
yps-3
and
yps 21:E-9
) were PCR amplified as labelled probes to identify nuclear DNA binding proteins which may influence phase-specific gene transcription. Protein-DNA interactions were assessed by Southwestern blot analysis in which sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated protein extracts from Y and M phases of the virulent G217B strain of
H. capsulatum
were visualized by their capability for in situ binding to the labelled 517-bp (G217B
yps-3
) or the 395-bp (G217B
yps 21:E-9
) putative 5′ regulatory regions. A 30-kDa nuclear protein unique to the M-phase extracts of the highly virulent G217B strain, but absent in the Y phase of the same organism, was identified. In contrast, the low-virulence, thermal-sensitive Downs strain of
H. capsulatum
lacked detectable p30 binding activity in either yeast- or mycelial phase extracts, regardless of the source of labelled probe (395-bp G217B
yps 21:E-9
probe or 512-bp
Hin
dIII-
Eco
RI-labelled Downs
yps21:E-9
). A decanucleotide motif, TCCTTTTTTT, was identified in the upstream regulatory regions of these
yps
genes, as well as in the putative α-tubulin promoter, and was conserved with 70 to 100% homology. This recognition sequence was sufficient for p30M binding with
32
P-labelled ligated oligonucleotides when used in the Southwestern assay. These findings describe the first nuclear DNA binding factor identified in
H. capsulatum
which binds to target sequences in a phase-specific manner, suggesting that p30M may govern aspects of gene transcription in this pathogenic fungus, in which a temperature-sensitive switch influences morphology and virulence.
Regulation of Macrophage Gene Expression byMycobacterium tuberculosis: Down-Regulation of Mitochondrial CytochromecOxidase
Authors: Ragno Silvia , Estrada-Garcia Iris , Butler Robert , Colston M. Joseph
Published: December 2019

We have investigated changes in gene expression in mouse peritoneal macrophages following infection with virulentMycobacterium tuberculosis. Using differential-display reverse transcription-PCR (RT-PCR), we have identified a gene that was markedly down-regulated within 6 h of infection and remained so for the duration of the experiment (5 days). On sequencing, this gene was found to encode the murine cytochromecoxidase subunit VIIc (COX VIIc). Down-regulation of COX VIIc duringM. tuberculosisinfection was confirmed by three independent techniques: limiting-dilution RT-PCR, RNase protection assay, and Northern analysis. Limiting-dilution RT-PCR and Northern analysis were also used to analyze the specificity of this regulation; heat-killedM. tuberculosis,Mycobacterium bovisBCG, and latex beads had no effect on expression of COX VIIc. Down-regulation of this enzyme was also confirmed by using adherent cells isolated from spleens ofM. tuberculosis-infected mice. These ex vivo macrophages showed apoptotic features, suggesting a possible involvement of cytochromecoxidase in the programmed cell death of the host cells.
Characterization of BrkA Expression in
Bordetella bronchiseptica
Authors: Rambow Amy A. , Fernandez Rachel C. , Weiss Alison A.
Published: December 2019

BrkA confers resistance to killing by complement in
Bordetella pertussis
. Complement resistance in
Bordetella bronchiseptica
was examined. Four
B. bronchiseptica
strains possessed the
brkA
gene; however, only three expressed the protein. Only the strain lacking BrkA was susceptible to complement. Introduction of the
B. pertussis brkA
gene restored BrkA expression to this strain but did not confer resistance.
brkA
was mutated in the strains that naturally expressed BrkA, and loss of BrkA did not confer sensitivity to complement. As a species,
B. bronchiseptica
is more resistant to complement than
B. pertussis
, and BrkA does not mediate resistance.
Vag8, a
Bordetella pertussis bvg
-Regulated Protein
Authors: Finn Theresa M. , Amsbaugh Diana F.
Published: December 2019

Bordetella pertussis
expresses a
bvg
-regulated 95-kDa protein, Vag8, encoded by
vag-8
. Southern blot analysis indicates that strains of
Bordetella bronchiseptica
and
Bordetella parapertussis
have DNA homologous to
vag-8
. Antiserum raised to a fusion of maltose binding protein to an N-terminal 60-kDa fragment of Vag8 recognizes the native 95-kDa protein in immunoblots of
B. pertussis
and
B. bronchiseptica
but not
B. parapertussis
. A 95-kDa protein-negative derivative of
B. pertussis
18323 containing a deletion of
vag-8
colonized mice as efficiently as the parent
B. pertussis
strain in a mouse aerosol model of pertussis.
Identification and Characterization of a 14-KilodaltonBrucella abortusProtein Reactive with Antibodies from Naturally and Experimentally Infected Hosts and T Lymphocytes from Experimentally Infected BALB/c Mice
Authors: Chirhart-Gilleland Rebecca L. , Kovach Michael E. , Elzer Philip H. , Jennings Stephen R. , Roop R. Martin
Published: December 2019

A low-molecular-weight recombinantBrucella abortusprotein reactive with antibodies from a variety of naturally and experimentally infected hosts and T lymphocytes from experimentally infected mice was identified and given the designation BA14K. The gene encoding BA14K was cloned and characterized, and the predicted amino acid sequence of this immunoreactive protein showed no significant homology with previously described proteins. Sequences homologous to the cloned fragment encoding BA14K were identified by Southern blot analysis of genomic DNAs from representatives of all of the currently recognizedBrucellaspecies. Studies employing BA14K should contribute to our efforts to better understand the antigenic specificity of protective immunity to brucellosis.
Catecholamine Oxidative Products, but Not Melanin, Are Produced byCryptococcus neoformansduring Neuropathogenesis in Mice
Authors: Liu Lide , Wakamatsu Kazumasa , Ito Shosuke , Williamson Peter R.
Published: December 2019

Melanin has been proposed as a virulence factor inCryptococcus neoformans, but its presence has not been shown unambiguously in vivo. Validated methods used previously to show production of cryptococcal eumelanin pigment in vitro (P. R. Williamson, K. Wakamatsu, and S. Ito, J. Bacteriol. 180:1570–1572, 1998) were used to assess for production of laccase-derived products in mouse brain of the Lacc+strains, 2E-TUC, H99 (serotype A), and ATCC 34873 (serotype D), and the Lacc−strain, 2E-TU. Pyrrole-2,3,5-tricarboxylic and pyrrole-2,3-dicarboxylic acid, specific degradation products of catecholamine derivatives such as melanin, were found in all Lacc+strains, but not in the Lacc−strain, 2E-TU. However, the presence of melanin pigment itself could not be demonstrated in the same cells. Lack of the specific degradation products aminohydroxyphenylalanine and aminohydroxyphenylethylamine in Lacc+strains upon hydriodic acid hydrolysis showed that pheomelanin was also not produced by the fungus in vivo. These are the first data to support the generation of catecholamine oxidation products byC. neoformansin vivo, but they do not support postenzymatic polymerization of these products to form typical eumelanin, as previously proposed.
Mutagenesis of Active-Site Histidines ofListeria monocytogenesPhosphatidylinositol-Specific Phospholipase C: Effects on Enzyme Activity and Biological Function
Authors: Bannam Trudi , Goldfine Howard
Published: December 2019

Listeria monocytogenes, a gram-positive facultative intracellular pathogen, produces two distinct phospholipases C. PC-PLC, encoded byplcB, is a broad-range phospholipase, whereas PI-PLC, encoded byplcA, is specific for phosphatidylinositol. It was previously shown that PI-PLC plays a role in efficient escape ofL. monocytogenesfrom the primary phagosome. To further understand the function of PI-PLC in intracellular growth, site-directed mutagenesis ofplcAwas performed. Two potential active-site histidine residues were mutated independently to alanine, serine, and phenylalanine. With the exception of the activity of the enzyme containing H38F, which was unstable, the PI-PLC enzyme activities of culture supernatants containing each mutant enzyme were <1% of wild-type activity. In addition, the levels of expression of the mutant PI-PLC proteins were equivalent to wild-type expression. Derivatives ofL. monocytogenescontaining these specificplcAmutations were found to have phenotypes similar to that of theplcAdeletion strain in an assay for escape from the primary vacuole, in intracellular growth in a murine macrophage cell line, and in a plaquing assay for cell-to-cell spread. Thus, catalytic activity of PI-PLC is required for all its intracellular functions.
Effects of Site-Directed Mutagenesis of
Escherichia coli
Heat-Labile Enterotoxin on ADP-Ribosyltransferase Activity and Interaction with ADP-Ribosylation Factors
Authors: A. Stevens Linda , Moss Joel , Vaughan Martha , Pizza Mariagrazia , Rappuoli Rino
Published: December 2019

Escherichia coli
heat-labile enterotoxin (LT), an oligomeric protein with one A subunit (LTA) and five B subunits, exerts its effects via the ADP-ribosylation of G
sα
, a guanine nucleotide-binding (G) protein that activates adenylyl cyclase. LTA also ADP-ribosylates simple guanidino compounds (e.g., arginine) and catalyzes its own auto-ADP-ribosylation. All LTA-catalyzed reactions are enhanced by ADP-ribosylation factors (ARFs), 20-kDa guanine nucleotide-binding proteins. Replacement of arginine-7 (R7K), valine-53 (V53D), serine-63 (S63K), valine 97 (V97K), or tyrosine-104 (Y104K) in LTA resulted in fully assembled but nontoxic proteins. S63K, V53D, and R7K are catalytic-site mutations, whereas V97K and Y104K are amino acid replacements adjacent to and outside of the catalytic site, respectively. The effects of mutagenesis were quantified by measuring ADP-ribosyltransferase activity (i.e., auto-ADP-ribosylation and ADP-ribosylagmatine synthesis) and interaction with ARF (i.e., inhibition of ARF-stimulated cholera toxin ADP-ribosyltransferase activity and effects of ARF on mutant auto-ADP-ribosylation). All mutants were inactive in the ADP-ribosyltransferase assay; however, auto-ADP-ribosylation in the presence of recombinant human ARF6 was detected, albeit much less than that of native LT (Y104K > V53D > V97K > R7K, S63K). Based on the lack of inhibition by free ADP-ribose, the observed auto-ADP-ribosylation activity was enzymatic and not due to the nonenzymatic addition of free ADP-ribose. V53D, S63K, and R7K were more effective than Y104K or V97K in blocking ARF stimulation of cholera toxin ADP-ribosyltransferase. Based on these data, it appears that ARF-binding and catalytic sites are not identical and that a region outside the NAD cleft may participate in the LTA-ARF interaction.
Differential Susceptibilities of
Anopheles albimanus
and
Anopheles pseudopunctipennis
to Infections with Coindigenous
Plasmodium vivax
Variants VK210 and VK247 in Southern Mexico
Authors: Gonzalez-Ceron Lilia , Rodriguez Mario H. , Nettel Jose C. , Villarreal Cuauhtemoc , Kain Kevin C. , Hernandez Juan E.
Published: December 2019

The susceptibilities to coindigenous
Plasmodium vivax
of colonized
Anopheles albimanus
and
Anopheles pseudopunctipennis
from southern Mexico were investigated by simultaneous feeding with infected blood obtained from patients. The genes encoding circumsporozoite protein variant types (VK210 and VK247) in blood samples were determined by PCR and oligonucleotide probe hybridization.
A. albimanus
was more susceptible to VK210, and
A. pseudopunctipennis
was more susceptible to VK247.
Antisense Inhibition of Expression of Cysteine Proteinases Affects
Entamoeba histolytica
-Induced Formation of Liver Abscess in Hamsters
Authors: Ankri Serge , Stolarsky Tamara , Bracha Rivka , Padilla-Vaca Felipe , Mirelman David
Published: December 2019

Trophozoites of virulent
Entamoeba histolytica
transfected with the antisense gene encoding cysteine proteinase 5 (CP5) have only 10% of the CP activity but retain their cytopathic activity on mammalian monolayers. In the present study we found that the transfected trophozoites with low levels of CP activity were incapable of inducing the formation of liver lesions in hamsters.
Soluble Tumor Necrosis Factor Alpha Receptors in Sera from Leprosy Patients
Authors: Munk Martin E. , Anding Peter , SchettinI Antonio P. M. , Cunha Maria da Graça S. , Kaufmann Stefan H. E.
Published: December 2019

Serum levels of soluble tumor necrosis factor alpha receptor I (sTNF-RI) were elevated in patients with lepromatous (LL) reactional-state type II leprosy, and sTNF-RII levels were increased in patients with full tuberculoid (TT) or LL type II leprosy. The sTNF-R in sera from patients with type II leprosy, but not other forms of leprosy, inhibited recombinant TNF cytolytic activities in vitro. This suggests that sTNF-R regulatory activities are partially impaired in patients with leprosy.
Glucose 6-Phosphate Dehydrogenase Is Required for
Salmonella typhimurium
Virulence and Resistance to Reactive Oxygen and Nitrogen Intermediates
Authors: Lundberg Brita E. , Wolf Richard E. , Dinauer Mary C. , Xu Yisheng , Fang Ferric C.
Published: December 2019

Salmonella typhimurium zwf
mutants lacking glucose 6-phosphate dehydrogenase (G6PD) activity have increased susceptibility to reactive oxygen and nitrogen intermediates as well as attenuated virulence in mice. Abrogation of the phagocyte respiratory burst oxidase during experimental infection with
zwf
mutant
Salmonella
causes a prompt restoration of virulence, while inhibition of inducible nitric oxide synthase results in delayed lethality. These observations suggest that G6PD-dependent bacterial antioxidant defenses play an important pathogenic role during early salmonellosis and additionally may help to antagonize NO-dependent antimicrobial mechanisms later in the course of infection.
Response to Reactive Nitrogen Intermediates inMycobacterium tuberculosis: Induction of the 16-Kilodalton α-Crystallin Homolog by Exposure to Nitric Oxide Donors
Authors: Garbe T. R. , Hibler N. S. , Deretic V.
Published: December 2019

In contrast to the apparent paucity ofMycobacterium tuberculosisresponse to reactive oxygen intermediates, this organism has evolved a specific response to nitric oxide challenge. Exposure ofM. tuberculosisto NO donors induces the synthesis of a set of polypeptides that have been collectively termed Nox. In this work, the most prominent Nox polypeptide, Nox16, was identified by immunoblotting and by N-terminal sequencing as the α-crystallin-related, 16-kDa small heat shock protein, sHsp16. A panel of chemically diverse donors of nitric oxide, with the exception of nitroprusside, induced sHsp16 (Nox16). Nitroprusside, a coordination complex of Fe2+with a nitrosonium (NO+) ion, induced a 19-kDa polypeptide (Nox19) homologous to the nonheme bacterial ferritins. We conclude that the NO response inM. tuberculosisis dominated by increased synthesis of the α-crystallin homolog sHsp16, previously implicated in stationary-phase processes and found in this study to be a majorM. tuberculosisprotein induced upon exposure to reactive nitrogen intermediates.
Effect of Melanin and Carotenoids of
Exophiala
(
Wangiella
)
dermatitidis
on Phagocytosis, Oxidative Burst, and Killing by Human Neutrophils
Authors: Schnitzler Norbert , Peltroche-Llacsahuanga Heidrun , Bestier Nicole , Zündorf Josef , Lütticken Rudolf , Haase Gerhard
Published: December 2019

The black yeast
Exophiala
(
Wangiella
)
dermatitidis
is an increasingly recognized pathogen and a leading cause of severe pheohyphomycosis. Melanin is thought to contribute to the virulence of
E. dermatitidis
. Whereas the synthesis and the redox properties of melanin have been studied intensively, the influence of melanin and carotenoids on the phagocytosis, the oxidative burst, and the killing of
E. dermatitidis
by human neutrophils has not been studied. To study their effects on these phenomena, we applied a combination of flow cytometry and a colony-count-dependent method. Using
E. dermatitidis
wild-type strain 8565 and several melanin-deficient mutants that have been described previously, we demonstrate that melanin prevents this pathogen from being killed in the phagolysosome of the neutrophils. Melanin did not influence the phagocytosis or the oxidative burst of the neutrophils involved. The carotenoids torulene and torularhodine were not found to contribute to the prevention of killing. The ability of
E. dermatitidis
to block the effects of the neutrophil oxidative burst may critically impair the potential of the host to sufficiently eliminate this fungal pathogen and thus may play an important role in the pathogenesis of phaeohyphomycosis.
Development and Characterization of a
Staphylococcus aureus
Nasal Colonization Model in Mice
Authors: Kiser Kevin B. , Cantey-Kiser Jean M. , Lee Jean C.
Published: December 2019

Staphylococcus aureus
nasal carriage is a risk factor for infection in humans, particularly in the hospital environment. Attenuation of carriage has proven effective in reducing the prevalence of infection in some high-risk groups. To study staphylococcal factors that influence nasal colonization, a mouse model of
S. aureus
nasal colonization was developed. Mice were inoculated intranasally with
S. aureus
Reynolds, and nasal carriage was evaluated by quantitating cultures of the nasal tissues from mice sacrificed at various time points after inoculation. The majority of mice inoculated with 10
8
CFU of
S. aureus
maintained nasal carriage for at least 20 days. Nasal colonization rates were similar for inbred (BALB/c and C57BL/6) and outbred (ICR) mice. Colonization was not affected by mouse passage of strain Reynolds. Lower inoculum doses (<10
7
CFU) resulted in reduced colonization after 7 days. However, mice given streptomycin in their drinking water developed long-term carriage of
S. aureus
, and they were colonized with inocula as low as 10
5
CFU. Nasal colonization was also established with two other
S. aureus
strains (one strain each of human and murine origins).
S. aureus
recovered from the nares of experimentally colonized mice expressed high levels of capsule, and the ability of a capsule-defective mutant to persist in the nares was reduced in comparison to that of the parent strain. This nasal colonization model should prove useful for studies of factors that mediate
S. aureus
colonization and for assessment of targets for antimicrobial intervention or vaccine development.
Clostridium botulinum
C2 Toxin Delays Entry into Mitosis and Activation of p34

cdc2

Kinase and cdc25-C Phosphatase in HeLa cells
Authors: Barth Holger , Klingler Manuela , Aktories Klaus , Kinzel Volker
Published: December 2019

The
Clostridium botulinum
C2 toxin ADP-ribosylates monomeric actin, thereby inducing disassembly of actin filaments, alteration of focal adhesions, and rounding of cells. After treatment with C2 toxin, cells stop to proliferate but remain viable for about 2 days. In view of reported correlations between the structure of the actin cytoskeleton and cell cycle transition, the effects of C2 toxin on the G
2
/M phase transition of the cell division cycle were studied. Since C2 toxin delayed entry into mitosis in HeLa cells, those enzymes which control entry into mitosis, the cyclin-dependent protein kinase mitosis-promoting factor (MPF) and the phosphatase cdc25-C were examined after treatment of synchronized cells with C2 toxin. MPF is composed of the regulatory cyclin B and the enzymatic p34

cdc2

kinase subunits. For its activation at the G
2
/M border, p34

cdc2

needs to be associated with cyclin B and additionally dephosphorylated at Tyr-15 by the specific phosphatase cdc25-C. Treatment of synchronized cells in S or G
2
phase with
C. botulinum
C2 toxin prevented p34

cdc2

protein kinase activation by inhibiting its tyrosine dephosphorylation at the G
2
/M border. Furthermore, the activity of cdc25-C phosphatase was decreased after treatment of cells with C2 toxin. Our results suggest that the prevented activation of the mitotic inducers p34

cdc2

kinase and cdc25-C phosphatase represents the final downstream events in the action of C2 toxin resulting in a G
2
phase cell cycle delay in synchronized HeLa cells.
The High-Pathogenicity Island of
Yersinia enterocolitica
Ye8081 Undergoes Low-Frequency Deletion but Not Precise Excision, Suggesting Recent Stabilization in the Genome
Authors: Bach Sandrine , Buchrieser Carmen , Prentice Michael , Guiyoule Annie , Msadek Tarek , Carniel Elisabeth
Published: December 2019

Highly pathogenic strains of
Yersinia pestis
,
Y. pseudotuberculosis
, and
Y. enterocolitica
are characterized by the possession of a pathogenicity island designated the high-pathogenicity island (HPI). This 35- to 45-kb island carries an iron uptake system named the yersiniabactin locus. While the HPIs of
Y. pestis
and
Y. pseudotuberculosis
are subject to high-frequency spontaneous deletion from the chromosome, we were initially unable to obtain HPI-deleted
Y. enterocolitica
1B isolates. In the present study, using a positive selection strategy, we identified three HPI-deleted mutants of
Y. enterocolitica
strain Ye8081. In these three independent clones, the chromosomal deletion was not limited to the HPI but encompassed a larger DNA fragment of approximately 140 kb. Loss of this fragment, which occurred at a frequency of approximately 5 × 10
−7
, resulted in the disappearance of several phenotypic traits, such as growth in a minimal medium, hydrolysis of
o
-nitrophenyl-β-
d
-thiogalactopyranoside, Tween esterase activity, and motility, and in a decreased virulence for mice. However, no precise excision of the Ye8081 HPI was observed. To gain more insight into the molecular basis for this phenomenon, the putative machinery of HPI excision in
Y. enterocolitica
was analyzed and compared to that in
Y. pseudotuberculosis
. We show that the probable reasons for failure of precise excision of the HPI of
Y. enterocolitica
Ye8081 are (i) the interruption of the P4-like integrase gene located close to its right-hand boundary by a premature stop codon and (ii) lack of conservation of 17-bp
att
-like sequences at both extremities of the HPI. These mutations may represent a process of HPI stabilization in the species
Y. enterocolitica
.
Acid Production by Vaginal Flora In Vitro Is Consistent with the Rate and Extent of Vaginal Acidification
Authors: Boskey E. R. , Telsch K. M. , Whaley K. J. , Moench T. R. , Cone R. A.
Published: December 2019

Perinatally, and between menarche and menopause, increased levels of estrogen cause large amounts of glycogen to be deposited in the vaginal epithelium. During these times, the anaerobic metabolism of the glycogen, by the epithelial cells themselves and/or by vaginal flora, causes the vagina to become acidic (pH ∼4). This study was designed to test whether the characteristics of acid production by vaginal flora in vitro can account for vaginal acidity. Eight vaginal
Lactobacillus
isolates from four species—
L. gasseri
,
L. vaginalis
,
L. crispatus
, and
L. jensenii
—acidified their growth medium to an asymptotic pH (3.2 to 4.8) that matches the range seen in the
Lactobacillus
-dominated human vagina (pH 3.6 to 4.5 in most women) (B. Andersch, L. Forssman, K. Lincoln, and P. Torstensson, Gynecol. Obstet. Investig. 21:19–25, 1986; L. Cohen, Br. J. Vener. Dis. 45:241–246, 1969; J. Paavonen, Scand. J. Infect. Dis. Suppl. 40:31–35, 1983; C. Tevi-Bénissan, L. Bélec, M. Lévy, V. Schneider-Fauveau, A. Si Mohamed, M.-C. Hallouin, M. Matta, and G. Grésenguet, Clin. Diagn. Lab. Immunol. 4:367–374, 1997). During exponential growth, all of these
Lactobacillus
species acidified their growth medium at rates on the order of 10
6
protons/bacterium/s. Such rates, combined with an estimate of the total number of lactobacilli in the vagina, suggest that vaginal lactobacilli could reacidify the vagina at the rate observed postcoitally following neutralization by the male ejaculate (W. H. Masters and V. E. Johnson, Human sexual response, p. 93, 1966). During bacterial vaginosis (BV), there is a loss of vaginal acidity, and the vaginal pH rises to >4.5. This correlates with a loss of lactobacilli and an overgrowth of diverse bacteria. Three BV-associated bacteria,
Gardnerella vaginalis
,
Prevotella bivia
, and
Peptostreptococcus anaerobius
, acidified their growth medium to an asymptotic pH (4.7 to 6.0) consistent with the characteristic elevated vaginal pH associated with BV. Together, these observations are consistent with vaginal flora, rather than epithelial cells, playing a primary role in creating the acidity of the vagina.
Quorum Sensing-Dependent Regulation and Blockade of Exoprotease Production in
Aeromonas hydrophila
Authors: Swift Simon , Lynch Martin J. , Fish Leigh , Kirke David F. , Tomás Juan M. , Stewart Gordon S. A. B. , Williams Paul
Published: December 2019

In
Aeromonas hydrophila
, the
ahyI
gene encodes a protein responsible for the synthesis of the quorum sensing signal
N
-butanoyl-
l
-homoserine lactone (C4-HSL). Inactivation of the
ahyI
gene on the
A. hydrophila
chromosome abolishes C4-HSL production. The exoprotease activity of
A. hydrophila
consists of both serine protease and metalloprotease activities; in the
ahyI
-negative strain, both are substantially reduced but can be restored by the addition of exogenous C4-HSL. In contrast, mutation of the LuxR homolog AhyR results in the loss of both exoprotease activities, which cannot be restored by exogenous C4-HSL. Furthermore, a substantial reduction in the production of exoprotease by the
ahyI
+
parent strain is obtained by the addition of
N
-acylhomoserine lactone analogs that have acyl side chains of 10, 12, or 14 carbons. The inclusion of
N
-(3-oxododecanoyl)-
l
-homoserine lactone or
N
-(3-oxotetradecanoyl)-
l
-homoserine lactone at 10 μM in overnight cultures of
A. hydrophila
abolishes exoprotease production in azocasein assays and reduces the activity of all the exoprotease species seen in zymograms.
Recognition of Fibronectin by
Penicillium marneffei
Conidia via a Sialic Acid-Dependent Process and Its Relationship to the Interaction Between Conidia and Laminin
Authors: Hamilton A. J. , Jeavons L. , Youngchim S. , Vanittanakom N.
Published: December 2019

Adhesion of
Penicillium marneffei
conidia to the extracellular matrix protein laminin via a sialic acid-dependent process has previously been demonstrated. This study describes the interaction of
P. marneffei
conidia with fibronectin and examines the relationship of this process to the recognition of laminin via conidia. Immunofluorescence microscopy demonstrated that fibronectin bound to the surface of conidia and to phialides, but not to hyphae, in a pattern similar to that reported for laminin. Conidia were able to bind to fibronectin immobilized on microtiter plates in a concentration-dependent manner. However, binding to fibronectin (at any given concentration of protein and conidia) was less than that to laminin under equivalent conditions. Soluble fibronectin and antifibronectin antibody inhibited adherence of conidia to fibronectin in the plate adherence assay; soluble laminin also caused pronounced inhibition. Various monosaccharides and several peptides had no effect on adherence to fibronectin. However,
N
-acetylneuraminic acid abolished adherence to fibronectin, indicating that the interaction was mediated through a sialic acid-dependent process; the latter parallels observations of laminin binding by conidia. Fibronectin binding (and binding of laminin) was considerably reduced by prolonged preincubation of conidia with chymotrypsin, suggesting the protein nature of the binding site. Conidia from older cultures were more adherent to both immobilized fibronectin and laminin than conidia from younger cultures. Ligand affinity binding demonstrated the presence of a 20-kDa protein with the ability to bind both fibronectin and laminin. There would therefore appear to be a common receptor for the binding of fibronectin and laminin on the surface of
P. marneffei
, and the interaction described here maybe important in mediating attachment of the fungus to host tissue.
A Two-Component Regulatory System, CsrR-CsrS, Represses Expression of Three
Streptococcus pyogenes
Virulence Factors, Hyaluronic Acid Capsule, Streptolysin S, and Pyrogenic Exotoxin B
Authors: Heath Andrew , DiRita Victor J. , Barg Neil L. , Engleberg N. Cary
Published: December 2019

Certain Tn
916
insertions in the chromosome of an M1-type, nonmucoid
Streptococcus pyogenes
isolate (MGAS166) were previously shown to result in stable mucoidy with increased expression of the capsular synthetic genes. The transposon insertions in these strains are directly upstream of an apparent operon encoding a two-component regulatory system, designated
csrR-csrS
. Compared with MGAS166, these mucoid mutants are more hemolytic and cause significantly more tissue damage in a murine model of skin infection. To extend these observations, we constructed an in-frame deletion in the gene encoding the response regulator,
csrR
, and we evaluated the expression of other known
S. pyogenes
virulence factors. We discovered that
csrR
mutants have enhanced transcription of
sagA
, a gene associated with streptolysin S (SLS) and
speB
, the gene encoding pyrogenic exotoxin B (SpeB). The mutants also express substantially higher SLS activity and SpeB antigen in late-exponential-phase cultures. There is no change in expression of
emm
,
scpA
,
sic
, or
cpa
(genes encoding other
S. pyogenes
virulence factors). CsrR
−
strains but not the wild-type parental strain produce necrotizing lesions in a mouse model of subcutaneous infection. A double mutant with deletions in both
csrR
and the capsular synthesis genes caused fewer and smaller necrotic skin lesions than the
csrR
mutants. However, this nonmucoid
csrR
strain was more likely than the wild type to yield necrotic lesions, suggesting that mucoidy contributes to virulence in this model of infection but that there are other
csrR
-regulated factors involved in the production of necrotic lesions.
Proteophosphoglycan, a Major Secreted Product of IntracellularLeishmania mexicanaAmastigotes, Is a Poor B-Cell Antigen and Does Not Elicit a Specific Conventional CD4+T-Cell Response
Authors: Aebischer Toni , Harbecke Dorothee , Ilg Thomas
Published: December 2019

Secreted and surface-exposed antigens of intracellular pathogens are thought to provide target structures for detection by the host immune system. The major secreted product of intracellularLeishmania mexicanaamastigotes, a proteophosphoglycan (aPPG), is known to contribute to the establishment of the parasitophorous vacuole and is able to activate complement. aPPG belongs to a novel class of serine- and threonine-richLeishmaniaproteins that are extensively modified by phosphodiester-linked phosphooligosaccharides and terminal mannooligosaccharides. Here we show that mice chronically infected withL. mexicanagenerally do not produce antibodies or Th cells specific for aPPG. Similarly, antibody titers are very low in mice vaccinated with aPPG, and specific CD4+T cells are undetectable. Comparative analyses of otherLeishmaniaglycoconjugates indicate thatL. mexicana-specific carbohydrate structures are poorly immunogenic in mice and that the proteophosphoglycan aPPG behaved immunologically like a carbohydrate. The latter observation is explained by the lack of induction of aPPG-specific CD4+T cells. In contrast, recombinant aPPG peptides stimulate CD4+T-cell responses and high titers of specific antibodies are found in the sera of mice vaccinated with these peptides. Native aPPG is highly resistant to proteinases and apparently cannot be degraded by macrophages. It is concluded that conventional CD4+T cells against the polypeptide backbone of aPPG are not induced because the molecule resists antigen processing due to its extensive and complex carbohydrate modification. The complex glycan chains of aPPG, which exhibit important biological functions for the parasite, may therefore also have evolved to evade detection by the immune system of the host organism.
Infection of Endothelial Cells with
Trypanosoma cruzi
Activates NF-κB and Induces Vascular Adhesion Molecule Expression
Authors: Huang Huan , Calderon Tina M. , Berman Joan W. , Braunstein Vicki L. , Weiss Louis M. , Wittner Murray , Tanowitz Herbert B.
Published: December 2019

Transcriptional activation of vascular adhesion molecule expression, a major component of an inflammatory response, is regulated, in part, by the nuclear factor-κB/Rel (NF-κB) family of transcription factors. We therefore determined whether
Trypanosoma cruzi
infection of endothelial cells resulted in the activation of NF-κB and the induction or increased expression of adhesion molecules. Human umbilical vein endothelial cells (HUVEC) were infected with trypomastigotes of the Tulahuen strain of
T. cruzi
. Electrophoretic mobility shift assays with an NF-κB-specific oligonucleotide and nuclear extracts from
T. cruzi
-infected HUVEC (6 to 48 h postinfection) detected two major shifted complexes. Pretreatment with 50× cold NF-κB consensus sequence abolished both gel-shifted complexes while excess SP-1 consensus sequence had no effect. These data indicate that nuclear extracts from
T. cruzi
-infected HUVEC specifically bound to the NF-κB consensus DNA sequence. Supershift analysis revealed that the gel-shifted complexes were comprised of p65 (RelA) and p50 (NF-κB1). Northern blot analyses demonstrated both the induction of vascular cell adhesion molecule 1 and E-selectin and the upregulation of intercellular adhesion molecule 1 mRNA in HUVEC infected with
T. cruzi
. Immunocytochemical staining confirmed adhesion molecule expression in response to
T. cruzi
infection. These findings are consistent with the hypothesis that the activation of the NF-κB pathway in endothelial cells associated with
T. cruzi
infection may be an important factor in the inflammatory response and subsequent vascular injury and endothelial dysfunction that lead to chronic cardiomyopathy.
Chlamydospore Formation in
Candida albicans
Requires the Efg1p Morphogenetic Regulator
Authors: Sonneborn Anja , Bockmühl Dirk P. , Ernst Joachim F.
Published: December 2019

Chlamydospore formation of the fungal pathogen
Candida albicans
was found to depend on the Efg1 protein, which regulates the yeast-hyphal transition. Isogenic mutants lacking
EFG1
or encoding T206A and T206E variants did not differentiate chlamydospores, while
cek1
,
cph1
, or
tpk2
mutations had no effect. Furthermore, filamentation of
efg1 cph1
double mutants in microaerophilic conditions suggests a novel Efg1p/Cph1p-independent filamentation pathway in
C. albicans.