bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

ABSTRACTListeria 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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACTSerum 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

ABSTRACT

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

ABSTRACTIn 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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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.