Biomedical subjects

Michael A Apicella

Publications and source records attributed to Michael A Apicella.

At least 19 recordsLinked to original sources

PubMed2006

Characterization of the OxyR regulon of Neisseria gonorrhoeae.

OxyR regulates the expression of the majority of H(2)O(2) responses in Gram-negative organisms. In a previous study we reported the OxyR-dependent derepression of catalase expression in the human pathogen Neisseria gonorrhoeae. In the present study we used microarray expression profiling of N. gonorrhoeae wild-type strain 1291 and an oxyR mutant strain to define the OxyR regulon. In addition to katA (encoding catalase), only one other locus displayed a greater than two-fold difference in expression in the wild type : oxyR comparison. This locus encodes an operon of two genes, a putative peroxiredoxin/glutaredoxin (Prx) and a putative glutathione oxidoreductase (Gor). Mutant strains were constructed in which each of these genes was inactivated. A previous biochemical study in Neisseria meningitidis had confirmed function of the glutaredoxin/peroxiredoxin. Assay of the wild-type 1291 cell free extract confirmed Gor activity, which was lost in the gor mutant strain. Phenotypic analysis of the prx mutant strain in H(2)O(2) killing assays revealed increased resistance, presumably due to upregulation of alternative defence mechanisms. The oxyR, prx and gor mutant strains were deficient in biofilm formation, and the oxyR and prx strains had decreased survival in cervical epithelial cells, indicating a key role for the OxyR regulon in these processes.

DNA-Binding Proteins↗

PubMed2006

Role of lgtC in resistance of nontypeable Haemophilus influenzae strain R2866 to human serum.

We are investigating a nontypeable Haemophilus influenzae (NTHI) strain, R2866, isolated from a child with meningitis. R2866 is unusually resistant to killing by normal human serum. The serum 50% inhibitory concentration (IC50) for this strain is 18%, approaching that of encapsulated H. influenzae. R3392 is a derivative of R2866 that was found to have increased sensitivity to human serum (IC50, 1.5%). Analysis of tetrameric repeat regions within lipooligosaccharide (LOS) biosynthetic genes in both strains indicated that the glycosyltransferase gene lgtC was out of frame ("off") in most colonies of R3392 but in frame with its start codon ("on") in most colonies of the parent. We sought antigenic and biochemical evidence for modification of the LOS structure. In a whole-cell enzyme-linked immunosorbent assay, strain R3392 displayed reduced binding of the Galalpha1,4Gal-specific monoclonal antibody 4C4. Mass spectrometry analysis of LOS from strain R2866 indicated that the primary oligosaccharide glycoform contained four heptose and four hexose residues, while that of R3392 contained four heptose and three hexose residues. We conclude that the R2866 lgtC gene encodes a galactosyltransferase involved in synthesis of the 4C4 epitope, as in other strains, and that expression of lgtC is associated with the high-level serum resistance that has been observed for this strain. This is the first description of the genetic basis of high-level serum resistance in NTHI, as well as the first description of LOS composition in an NTHI strain for which the complete genome sequence has been determined.

Animals↗

PubMed2006

Neisseria gonorrhoeae delays the onset of apoptosis in polymorphonuclear leukocytes.

Neisseria gonorrhoeae (gonococcus) infection results in recruitment of polymorphonuclear leukocytes (PMNs) to the urethral lumen. Recent work from our laboratory demonstrated that N. gonorrhoeae resists killing and replicates within PMNs. In this study, we examined the effect of gonococci on PMN viability. Using both transmission electron microscopy and light microscopy, we observed nuclear condensation after 6 h in PMNs that were resting or challenged with opsonized zymosan particles (OPZ). In contrast, N. gonorrhoeae delayed nuclear condensation in PMNs for 12 h (13% apoptotic PMNs vs. 90% for resting and 94% for OPZ-stimulated PMNs). Additionally, DNA fragmentation was reduced in PMNs challenged with gonococci for 12 h (28% apoptosis vs. 52% for resting and 98% for OPZ-stimulated PMNs). However, 74% of PMNs challenged with gonococci had condensed nuclei and 67% had fragmented DNA after 24 h. Caspase activity (total caspase, caspase-3/7, caspase-9) was reduced at 4 h and mitochondrial integrity was preserved at 2 h in PMNs challenged with N. gonorrhoeae. Quantitative reverse transcription polymerase chain reaction demonstrated that mRNA levels of X-IAP and cIAP-2 remained high after challenge with gonococci, but were downregulated in OPZ-stimulated PMNs. Collectively, these findings demonstrate that N. gonorrhoeae delayed apoptosis in PMNs, perhaps as a strategy to allow intracellular replication.

Apoptosis↗

PubMed2006

Dynamics of dendritic cell migration and the subsequent induction of protective immunity in the lung after repeated airway challenges by nontypeable Haemophilus influenzae outer membrane protein.

To determine the dynamics of dendritic cell (DCs) migration and their role in recurrent infections by nontypeable Haemophilus influenzae (NTHi), the migration of mature DC into pulmonary lymph nodes (LN) and the development of a P6-specific immune response and bacterial clearance in the lung were examined after repeated airway challenges with outer membrane protein (OMP) at 1-week intervals in mice. Although the migration of mature DC into the pulmonary LN is attenuated after repeated airway challenge with OMP, the in vitro P6-specific T cell proliferation in the cultured pulmonary LN cells was enhanced and was subsequently linked to the development of P6-specific IgA production and the development of protective immunity in the airway of mice.

Administration, Inhalation↗

PubMed2006

Haemophilus influenzae forms biofilms on airway epithelia: implications in cystic fibrosis.

RATIONALE: Nontypeable Haemophilus influenzae (NTHi) commonly infects patients with cystic fibrosis (CF), especially early in childhood. Bacteria biofilms are increasingly recognized as contributing to bacterial persistence and disease pathogenesis in CF. OBJECTIVES: This study investigated ability of NTHi to form biofilms and its impact on airway epithelia using in vivo and in vitro analyses. METHODS: We evaluated bronchoalveolar lavage fluid from young patients with CF for evidence of NTHi biofilms. To further investigate the pathogenesis of NTHi in respiratory infections, we developed a novel in vitro coculture model of NTHi biofilm formation on polarized human airway epithelial cells grown at the air-liquid interface. MEASUREMENTS AND MAIN RESULTS: In bronchoalveolar lavage fluid samples from young, asymptomatic patients with CF, we found morphologic evidence suggestive of NTHi biofilm formation. In addition, 10 clinical NTHi isolates from patients with CF formed biofilms on plastic surfaces. NTHi formed biofilms on the apical surface of cultured airway epithelia. These biofilms exhibited decreased susceptibility to antibiotics and were adherent to epithelial surfaces. Airway epithelial cells remained viable throughout 4 d of coculture, and responded to NTHi with nuclear factor-kappaB signaling, and increased chemokine and cytokine secretion. CONCLUSIONS: NTHi formed adherent biofilms on the apical surface airway epithelia with decreased susceptibility to antibiotics, and respiratory cells exhibited inflammatory and host defense responses-evidence of a dynamic host-pathogen interaction. The data presented here have implications both for understanding early CF lung disease pathogenesis and for the treatment of early, asymptomatic colonization of patients with CF with H. influenzae.

Biofilms↗

PubMed2006

PerR controls Mn-dependent resistance to oxidative stress in Neisseria gonorrhoeae.

In previous studies it has been established that resistance to superoxide by Neisseria gonorrhoeae is dependent on the accumulation of Mn(II) ions involving the ABC transporter, MntABC. A mutant strain lacking the periplasmic binding protein component (MntC) of this transport system is hypersensitive to killing by superoxide anion. In this study the mntC mutant was found to be more sensitive to H2O2 killing than the wild-type. Analysis of regulation of MntC expression revealed that it was de-repressed under low Mn(II) conditions. The N. gonorrhoeae mntABC locus lacks the mntR repressor typically found associated with this locus in other organisms. A search for a candidate regulator of mntABC expression revealed a homologue of PerR, a Mn-dependent peroxide-responsive regulator found in Gram-positive organisms. A perR mutant expressed more MntC protein than wild-type, and expression was independent of Mn(II), consistent with a role for PerR as a repressor of mntABC expression. The PerR regulon of N. gonorrhoeae was defined by microarray analysis and includes ribosomal proteins, TonB-dependent receptors and an alcohol dehydrogenase. Both the mntC and perR mutants had reduced intracellular survival in a human cervical epithelial cell model.

Bacterial Proteins↗

PubMed2006

Neisseria gonorrhoeae PLD directly interacts with Akt kinase upon infection of primary, human, cervical epithelial cells.

Neisseria gonorrhoeae secrets a phospholipase D (NgPLD), which augments complement receptor 3 (CR3)-mediated invasion of cervical epithelial cells. To elucidate the signalling pathways triggered with gonococcus CR3-engagement and the putative function of NgPLD in these events, we analysed the contribution of the phosphoinositide-Akt pathway to cervical infection. Our data indicated that Akt plays a critical role in cervical infection. Inhibition of myosin light chain kinase, PtdIns(4,5)P2, and Akt functions resulted in decreased gonococcus invasion of primary, human, cervical epithelial cells as well as Akt kinase activity. Akt activity was similarly impaired when cervical cells were challenged with NgPLD-mutant gonococci. Conversely, the PI3-kinase inhibitor, LY294002, enhanced gonococcal invasion of, and Akt activity within, primary cervical cells. We demonstrated that NgPLD directly binds to the Akt PH domain and can compete with a natural Akt ligand, PtdIns(3,4,5)P3, for Akt binding. Collectively, our data suggested that NgPLD augments gonococcus invasion of cervical epithelia by interacting with Akt kinase in a PI3-kinase-independent manner, which results in subversion of normal cervical cell signalling.

Binding Sites↗

PubMed2006

Basis for the failure of Francisella tularensis lipopolysaccharide to prime human polymorphonuclear leukocytes.

Francisella tularensis is the intracellular gram-negative coccobacillus that causes tularemia, and its virulence and infectiousness make it a potential agent of bioterrorism. Previous studies using mononuclear leukocytes have shown that the lipopolysaccharide (LPS) of F. tularensis is neither a typical proinflammatory endotoxin nor an endotoxin antagonist. This inertness suggests that F. tularensis LPS does not bind host LPS-sensing molecules such as LPS-binding protein (LBP). Using priming of the polymorphonuclear leukocyte (PMN) oxidase as a measure of endotoxicity, we found that F. tularensis live vaccine strain LPS did not behave like either a classic endotoxin or an endotoxin antagonist in human PMNs, even when the concentration of LBP was limiting. Furthermore, F. tularensis LPS did not compete with a radiolabeled lipooligosaccharide from Neisseria meningitidis for binding to LBP or to the closely related PMN granule protein, bactericidal/permeability-increasing protein. Our results suggest that the inertness of F. tularensis LPS and the resistance of F. tularensis to oxygen-independent PMN killing may result from the inability of F. tularensis LPS to be recognized by these important LPS-sensing molecules of the innate immune system.

Acute-Phase Proteins↗

PubMed2006

Microbial pattern recognition receptors mediate M-cell uptake of a gram-negative bacterium.

The receptors involved in the sampling of particulate microbial antigens by the gut are largely unknown. Here we demonstrate for the first time in an in vitro M-cell model and in situ in isolated murine intestinal segments that the receptors TLR-4, PAF-R, and alpha5beta1 integrin are all involved in mediating bacterial uptake associated with transcytosis. The pattern of expression of TLR-4 and alpha5beta1 integrin differed between M cells and enterocytes. There was increased apical expression of TLR-4 in M-cell cultures, and it was present on the apical surface of murine M cells but not enterocytes in situ. In contrast, PAF-R was expressed equally by both cell types in vitro and was abundantly expressed throughout the intestinal epithelium. Inhibition of TLR-4 and PAF-R, but not TLR-2, reduced gram-negative bacterial uptake by both cell types, whereas inhibition of the apically expressed alpha5beta1 integrin significantly reduced the ability of M cells to translocate bacteria. Hence, the involvement of each receptor was dependent not only on differences in the level of receptor expression but the cellular localization. Using bacteria that had mutations that affected the bacterial lipooligosaccharide structure indicated that the oligosaccharide moiety was important in bacterial uptake. Taken together, the data suggest that pathogen-associated molecular pattern interactions with pattern recognition receptors are key factors in M-cell recognition of intestinal antigens for mucosal immune priming.

Animals↗

PubMed2006

Defenses against oxidative stress in Neisseria gonorrhoeae: a system tailored for a challenging environment.

Neisseria gonorrhoeae is a host-adapted pathogen that colonizes primarily the human genitourinary tract. This bacterium encounters reactive oxygen and reactive nitrogen species as a consequence of localized inflammatory responses in the urethra of males and endocervix of females and also of the activity of commensal lactobacilli in the vaginal flora. This review describes recent advances in the understanding of defense systems against oxidative stress in N. gonorrhoeae and shows that while some of its defenses have similarities to the paradigm established with Escherichia coli, there are also some key differences. These differences include the presence of a defense system against superoxide based on manganese ions and a glutathione-dependent system for defense against nitric oxide which is under the control of a novel MerR-like transcriptional regulator. An understanding of the defenses against oxidative stress in N. gonorrhoeae and their regulation may provide new insights into the ways in which this bacterium survives challenges from polymorphonuclear leukocytes and urogenital epithelial cells.

Catalase↗

PubMed2006

Francisella tularensis: taxonomy, genetics, and Immunopathogenesis of a potential agent of biowarfare.

Tularemia is a zoonosis of humans caused by infection with the facultative intracellular bacterium Francisella tularensis. Interest in F. tularensis has increased markedly in the past few years because of its potential use as an agent of bioterrorism. Five subspecies of this organism are found in the Northern hemisphere, but only F. tularensis subsp. tularensis and subsp. holarctica cause disease in humans. This review summarizes what is known about the pathogenesis of tularemia with a focus on bacterial surface components such as lipopolysaccharide and capsule as well as information obtained from the F. tularensis subsp. tularensis SCHU S4 genome. In particular, the mechanisms of action of recently identified virulence factors are discussed in the context of bacterial replication in macrophages and manipulation of the host inflammatory response. Throughout this report, shared and unique features of F. tularensis subsp. tularensis, subsp. holarctica, and subsp. novicida are discussed.

Animals↗

PubMed2005

Lipopolysaccharide deacylation by an endogenous lipase controls innate antibody responses to Gram-negative bacteria.

T cell-independent type 1 agonists such as Gram-negative bacterial lipopolysaccharides can stimulate B lymphocytes to proliferate and produce antibodies by signaling through Toll-like receptors. This phenomenon is well established in vitro, yet polyclonal B cell responses after bacterial infection in vivo are often weak and short-lived. We show here that B cell proliferation and polyclonal antibody production in response to Gram-negative bacterial infection are modulated by acyloxyacyl hydrolase, a host enzyme that deacylates bacterial lipopolysaccharides. Deacylation of lipopolysaccharide occurred over several days, allowing lipopolysaccharide to act as an innate immune stimulant yet limiting the eventual amount of B cell proliferation and polyclonal antibody production. Control of lipopolysaccharide activation by acyloxyacyl hydrolase indicates that mammals can regulate immune responses to bacterial infection by chemical modification of a Toll-like receptor agonist.

Acetylation↗

PubMed2005

Carbohydrate moieties as vaccine candidates.

Carbohydrate epitopes or glycotopes are structurally diverse, occur in a variety of chemical contexts, and are present on the surfaces of cells in the body and on the surfaces of pathogens. These various structures and modes of presentation affect how they are perceived and processed by the body and dictate the outcome of the immune response directed against them. This review focuses on mechanisms of carbohydrate immunity, with an emphasis on carbohydrate vaccines that have been or are being developed for protection against encapsulated bacterial pathogens. We discuss the cellular basis of carbohydrate immunity, newly identified glycotope processing pathways and recognition capabilities, and the synthetic and microarray technologies that are being developed that will permit new experimental approaches to carbohydrate vaccine development and the exploration of the interaction of the immune system with self and nonself glycans.

Antibodies, Bacterial↗

PubMed2005

NmlR of Neisseria gonorrhoeae: a novel redox responsive transcription factor from the MerR family.

A MerR-like regulator (NmlR -Neisseria merR-like Regulator) identified in the Neisseria gonorrhoeae genome lacks the conserved cysteines known to bind metal ions in characterized proteins of this family. Phylogenetic analysis indicates that NmlR defines a subfamily of MerR-like transcription factors with a distinctive pattern of conserved cysteines within their primary structure. NmlR regulates itself and three other genes in N. gonorrhoeae encoding a glutathione-dependent dehydrogenase (AdhC), a CPx-type ATPase (CopA) and a thioredoxin reductase (TrxB). An nmlR mutant lacked the ability to survive oxidative stress induced by diamide and cumene hydroperoxide. It also had > 50-fold lower NADH-S-nitrosoglutathione oxidoreductase activity consistent with a role for AdhC in protection against nitric oxide stress. The upstream sequences of the NmlR regulated genes contained typical MerR-like operator/promoter arrangements consisting of a dyad symmetry located between the -35 and -10 elements of the target genes. The NmlR target operator/promoters were cloned into a beta-galactosidase reporter system and promoter activity was repressed by the introduction of NmlR in trans. Promoter activity was activated by NmlR in the presence of diamide. Under metal depleted conditions NmlR did not repress P(AdhC) (or P(CopA)) promoter activity, but this was reversed in the presence of Zn(II), indicating repression was Zn(II)-dependent. Analysis of mutated promoters lacking the dyad symmetry revealed constitutive promoter activity which was independent of NmlR. Gel shift assays further confirmed that NmlR bound to the target promoters possessing the dyad symmetry. Site-directed mutagenesis of the four NmlR cysteine residues revealed that they were essential for activation of gene expression by NmlR.

Amino Acid Sequence↗

PubMed2005

Ultrastructural analysis of the pathogenesis of Neisseria gonorrhoeae endometrial infection.

We have studied gonococcal infection in human endometrium organ culture and in human primary endometrial epithelial cells using various microscopic techniques including scanning electron microscopy, transmission electron microscopy, bright field light microscopy and laser scanning confocal microscopy. Here we describe the interactions between Neisseria gonorrhoeae and human endometrial luminal epithelial cells at the ultrastructural levels. N. gonorrhoeae attached to cilia but were not observed associated with the plasma membrane of ciliated epithelial cells or internalized into ciliated epithelial cells. N. gonorrhoeae could be found in intracellular vacuoles in secretory epithelial cells. N. gonorrhoeae have diverse interactions with endometrial epithelium. These include intimate association and colocalization with asialoglycoprotein receptor (ASGP-R) and CEACAM, lamellipodia and ruffle formation and colocalization with CR3, and microvillus engagement. These studies indicate that N. gonorrhoeae utilize multiple mechanisms to associate with endometrial epithelial cells and can associate with both ciliated and secretory cells. This diversity is consistent with a role of the endometrium as a transition zone between frequently asymptomatic cervical gonorrhoea and symptomatic pelvic inflammatory disease.

Antigens, CD↗

PubMed2005

I-domain-containing integrins serve as pilus receptors for Neisseria gonorrhoeae adherence to human epithelial cells.

Two pilus receptors are identified for the pathogenic Neisseria, CD46 and complement receptor 3. An intimate association between the asialoglycoprotein receptor and gonococcal lipooligosaccharide mediates invasion of primary, male urethral epithelial cells (UECs); however, studies to identify pilus receptors on these cells have not been performed. Based on our previous studies we reasoned that the I-domain-containing (IDC), alpha(1)- and alpha(2)-integrins might serve as pilus receptors on UECs and on urethral tissue. Confocal microscopy revealed colocalization of pilus with alpha(1) and alpha(2) integrins on UECs and tissue. We found that recombinant I-domain and antibodies directed against the alpha(1)- and alpha(2)-integrins inhibited gonococcal association with UECs and with immortal cell lines of variable origin. Gonococcus-integrin colocalization occurred at early time points post infection, but this interaction dissociated with extended infection. Similarly, Western Blot analyses revealed that gonococcal pilin coimmunoprecipitates with alpha(1)- and alpha(2)-integrins. However, studies performed in parallel and that were designed to capture CD46-pilus immune complexes indicated that a CD46-pilus interaction did not occur. Collectively, these data suggest that while CD46 might be able to bind gonococcal pilus, IDC integrins are preferentially used as the initial docking site for gonococci on UECs, on urethral tissue and on some immortal cell lines.

Asialoglycoprotein Receptor↗

PubMed2005

Azurin of pathogenic Neisseria spp. is involved in defense against hydrogen peroxide and survival within cervical epithelial cells.

Laz, a lipid-modified azurin of the human pathogens Neisseria gonorrhoeae and Neisseria meningitidis, is involved in defense against oxidative stress and copper toxicity; laz mutant strains are hypersensitive to hydrogen peroxide and copper. The N. gonorrhoeae laz mutant also has decreased survival in an ex vivo primary human ectocervical epithelial assay.

Azurin↗

PubMed2005

Interactions of Neisseria gonorrhoeae with adherent polymorphonuclear leukocytes.

Neisseria gonorrhoeae causes severe exudative urethritis. The exudates from infected individuals contain large numbers of polymorphonuclear leukocytes (PMN) with ingested gonococci. The fate of N. gonorrhoeae within PMN has been a topic of debate for years. In this study, we examined the interactions of N. gonorrhoeae with PMN adherent to surfaces as a system that better models events during clinical disease. Using chemiluminescence to measure reactive oxygen species (ROS), we found that N. gonorrhoeae stimulated PMN to produce a respiratory burst. Different kinetics were seen when PMN were stimulated with opsonized zymosan particles. In addition, ROS were produced predominantly inside the PMN in response to gonococci. Laser scanning confocal microscopy and transmission electron microscopy showed that N. gonorrhoeae rapidly associated with PMN under these experimental conditions and was internalized. Some gonococci were cleared in the first 30 to 60 min after phagocytosis, but a majority of the population persisted for 6 h after phagocytosis. Quantification of viable organisms showed that a significant portion of the population resisted killing. The viability of this subpopulation remained unchanged for 2 h after phagocytosis. A significant increase of viable gonococci from 1 to 6 h was also observed, suggesting intracellular replication. Four different N. gonorrhoeae strains demonstrated the same capacity to resist PMN-mediated killing, whereas Escherichia coli was rapidly killed by PMN under the same conditions. Taken together, these findings suggest that a subpopulation of N. gonorrhoeae resists killing and replicates within PMN phagosomes in spite of NADPH oxidase activation.

Cell Adhesion↗