Margaret R. Ketterer

Binding of the non-typeable Haemophilus influenzae lipooligosaccharide to the PAF receptor initiates host cell signalling.

Non‐typeable Haemophilus influenzae (NTHi) invades host cells by binding of the platelet‐activating factor (PAF) receptor via lipooligosaccharide (LOS) glycoforms containing phosphorylcholine (ChoP). The effect of NTHi infection on host cell signalling and its role in NTHi invasion was examined. The infection of human bronchial epithelial cells with NTHi 2019 increased cytosolic Ca2+ levels, and the invasion of bronchial cells by NTHi 2019 was inhibited by pretreatment with the cell‐permeant intracellular Ca2+ chelator BAPTA‐AM (P = 0.022) or thapsigargin (P = 0.016). Cytosolic inositol phosphate (IP) levels were also increased after infection with NTHi 2019 (P < 0.001), but not after infection with isogenic mutants expressing altered LOS glycoforms lacking ChoP. PAF receptor antagonist reduced NTHi 2019‐stimulated IP production in a dose‐dependent manner. NTHi 2019 invasion was inhibited by pertussis toxin (PTX) and the phosphatidylinositol‐3‐kinase inhibitors wortmannin and LY294002. The less invasive strain NTHi 7502 also initiated IP production, but was unaffected by PAF receptor antagonist or PTX. These data demonstrate that the binding of the PAF receptor by NTHi initiates receptor coupling to a PTX‐sensitive heterotrimeric G protein complex, resulting in a multifactorial host cell signal cascade and bacterial invasion. Moreover, the data suggest that NTHi strains initiate cell signalling and invade by different mechanisms, and that invasion mediated by PAF receptor activation is more efficient than macropinocytosis.

Cutting Edge: Mutation of Francisella tularensis mviN Leads to Increased Macrophage Absent in Melanoma 2 Inflammasome Activation and a Loss of Virulence

The mechanisms by which the intracellular pathogen Francisella tularensis evades innate immunity are not well defined. We have identified a gene with homology to Escherichia coli mviN, a putative lipid II flippase, which F. tularensis uses to evade activation of innate immune pathways. Infection of mice with a F. tularensis mviN mutant resulted in improved survival and decreased bacterial burdens compared to infection with wild-type F. tularensis. The mviN mutant also induced increased absent in melanoma 2 inflammasome-dependent IL-1β secretion and cytotoxicity in macrophages. The compromised in vivo virulence of the mviN mutant depended upon inflammasome activation, as caspase 1- and apoptosis-associated speck-like protein containing a caspase recruitment domain-deficient mice did not exhibit preferential survival following infection. This study demonstrates that mviN limits F. tularensis-induced absent in melanoma 2 inflammasome activation, which is critical for its virulence in vivo.

Gonococcal Cervicitis: A Role for Biofilm in Pathogenesis

Neisseria gonorrhoeae forms a biofilm in flow cells on glass coverslips as well as on primary cervical epithelial cells. Electron microscopic studies of cervical biopsy specimens from 10 patients with culture-proven N. gonorrhoeae infection revealed evidence of biofilm formation in 3 of the biopsy specimens. These biofilms showed gonococci in networks of bacterial membrane within the biofilm structure. This finding was also observed in biofilms formed over glass cover slips and after infection of primary cervical tissue in vitro. The importance of membranous networks in Neisseria biofilm formation was demonstrated with N. gonorrhoeae strain 1291-msbB, which shows a markedly decreased ability to bleb. This mutant formed significantly less biofilm over glass surfaces and cervical epithelial cells, and complementation showed reversion to wild-type biofilms. Gonoccal biofilms, as part of the cervical infection, may be involved in the mechanisms by which asymptomatic infections, persistence, and increased antibiotic resistance occur.

The Composition and Metabolic Phenotype of Neisseria gonorrhoeae Biofilms.

Neisseria gonorrhoeae has been shown to form biofilms during cervical infection. Thus, biofilm formation may play an important role in the infection of women. The ability of N. gonorrhoeae to form membrane blebs is crucial to biofilm formation. Blebs contain DNA and outer membrane structures, which have been shown to be major constituents of the biofilm matrix. The organism expresses a DNA thermonuclease that is involved in remodeling of the biofilm matrix. Comparison of the transcriptional profiles of gonococcal biofilms and planktonic runoff indicate that genes involved in anaerobic metabolism and oxidative stress tolerance are more highly expressed in biofilm. The expression of aniA, ccp, and norB, which encode nitrite reductase, cytochrome c peroxidase, and nitric oxide reductase respectively, is required for mature biofilm formation over glass and human cervical cells. In addition, anaerobic respiration occurs in the substratum of gonococcal biofilms and disruption of the norB gene required for anaerobic respiration, results in a severe biofilm attenuation phenotype. It has been demonstrated that accumulation of nitric oxide (NO) contributes to the phenotype of a norB mutant and can retard biofilm formation. However, NO can also enhance biofilm formation, and this is largely dependent on the concentration and donation rate or steady-state kinetics of NO. The majority of the genes involved in gonococcal oxidative stress tolerance are also required for normal biofilm formation, as mutations in the following genes result in attenuated biofilm formation over cervical cells and/or glass: oxyR, gor, prx, mntABC, trxB, and estD. Overall, biofilm formation appears to be an adaptation for coping with the environmental stresses present in the female genitourinary tract. Therefore, this review will discuss the studies, which describe the composition and metabolic phenotype of gonococcal biofilms.

Macropinocytosis as a Mechanism of Entry into Primary Human Urethral Epithelial Cells by Neisseria gonorrhoeae

ABSTRACT Gonococcal entry into primary human urethral epithelial cells (HUEC) can occur by macropinocytosis. Scanning and transmission electron microscopy revealed lamellipodia surrounding gonococci, and confocal laser scanning microscopy analysis showed organisms colocalized with Mr 70,000 fluorescein isothiocyanate-labeled dextran within the cells. Phosphoinositide 3-kinase inhibitors and an actin polymerization inhibitor prevented macropinocytic entry of gonococci into HUEC.

Comparative analyses of proteins from Haemophilus influenzae biofilm and planktonic populations using metabolic labeling and mass spectrometry

BackgroundNon-typeable H. influenzae (NTHi) is a nasopharyngeal commensal that can become an opportunistic pathogen causing infections such as otitis media, pneumonia, and bronchitis. NTHi is known to form biofilms. Resistance of bacterial biofilms to clearance by host defense mechanisms and antibiotic treatments is well-established. In the current study, we used stable isotope labeling by amino acids in cell culture (SILAC) to compare the proteomic profiles of NTHi biofilm and planktonic organisms. Duplicate continuous-flow growth chambers containing defined media with either “light” (L) isoleucine or “heavy” (H) 13C6-labeled isoleucine were used to grow planktonic (L) and biofilm (H) samples, respectively. Bacteria were removed from the chambers, mixed based on weight, and protein extracts were generated. Liquid chromatography-mass spectrometry (LC-MS) was performed on the tryptic peptides and 814 unique proteins were identified with 99% confidence.ResultsComparisons of the NTHi biofilm to planktonic samples demonstrated that 127 proteins showed differential expression with p-values ≤0.05. Pathway analysis demonstrated that proteins involved in energy metabolism, protein synthesis, and purine, pyrimidine, nucleoside, and nucleotide processes showed a general trend of downregulation in the biofilm compared to planktonic organisms. Conversely, proteins involved in transcription, DNA metabolism, and fatty acid and phospholipid metabolism showed a general trend of upregulation under biofilm conditions. Selected reaction monitoring (SRM)-MS was used to validate a subset of these proteins; among these were aerobic respiration control protein ArcA, NAD nucleotidase and heme-binding protein A.ConclusionsThe present proteomic study indicates that the NTHi biofilm exists in a semi-dormant state with decreased energy metabolism and protein synthesis yet is still capable of managing oxidative stress and in acquiring necessary cofactors important for biofilm survival.

The msbB Mutant of Neisseria meningitidis Strain NMB Has a Defect in Lipooligosaccharide Assembly and Transport to the Outer Membrane

ABSTRACT A deletion-insertion mutation in msbB, a gene that encodes a lipid A acyltransferase, was introduced into encapsulated Neisseria meningitidis serogroup B strain NMB and an acapsular mutant of the same strain. These mutants were designated NMBA11K3 and NMBA11K3cap-, respectively. Neither lipooligosaccharide (LOS) nor lipid A could be isolated from NMBA11K3 although a number of techniques were tried, but both were easily extracted from NMBA11K3cap-. Immunoelectron microscopy using monoclonal antibody (MAb) 6B4, which recognizes the terminal Galβ1-4GlcNAc of LOS, demonstrated that NMB, NMBcap-, and NMBA11K3cap- expressed LOS circumferentially, while MAb 6B4 did not bind to the surface of NMBA11K3. However, cytoplasmic staining of NMBA11K3 with MAb 6B4 was a consistent observation. Mass-spectrometric analyses demonstrated that the relative amounts of the lipid A-specific C12:0 3-OH and C14:0 3-OH present in the membrane preparations (MP) from NMBA11K3 were substantially decreased (25- and 23-fold, respectively) compared to the amount in MP from its parent strain, NMB. Western blot analyses of MP from NMBA11K3 demonstrated that the levels of porin in the outer membrane of NMBA11K3 were also substantially decreased. These studies suggest that the lipid A acylation defect in encapsulated NMBA11K3 influences the assembly of the lipid A and consequently the incorporation of porin in the outer membrane.

A Pilus-Deficient Mutant of Haemophilus ducreyi Is Virulent in the Human Model of Experimental Infection

Haemophilus ducreyi expresses fine tangled pili, which are composed predominantly of a major subunit (FtpA). Confocal microscopy showed that an FtpA-specific monoclonal antibody bound to bacteria in biopsy samples obtained from infected human volunteers. To test the role of pili in pathogenesis, an isogenic mutant (35000HP-SMS1) was constructed by insertionally inactivating ftpA. 35000HP-SMS1 did not express FtpA and was nonpiliated but was otherwise identical to its parent, 35000HP. Seven healthy adults were challenged on the upper arm with the isogenic isolates in a double-blinded, escalating dose-response study. Sites inoculated with the mutant produced papules and pustules at rates similar to the rates observed at sites inoculated with the parent. The recovery rate of H. ducreyi from cultures and the histopathology of biopsy samples obtained from pustules inoculated with 35000HP or 35000HP-SMS1 were similar. Although pili are expressed in vivo, FtpA is not required for pustule formation in the human challenge model.

Recombinant truncated AniA of pathogenic Neisseria elicits a non-native immune response and functional blocking antibodies.

AniA of the pathogenic Neisseria is glycosylated in its C-terminal repeat region by the pilin glycosylation (pgl) pathway. AniA appears to be unique among bacterial nitrite reductases as it contains an N-terminal extension that includes a lipid modification site as well as a C-terminal extension that is glycosylated. Immunising with various glycoforms of the AniA protein demonstrated a strong humoral immune response to the basal monosaccharide. In addition, when animals were immunised with a truncated form of AniA, completely lacking the glycosylated C-terminal region, the antibody response was directed against AniA regardless of the glycosylation state of the protein. Immuno-SEM confirmed that AniA is expressed on the cell surface in Neisseria gonorrhoeae. Antisera generated against a truncated, non-glycosylated, recombinant form of the AniA protein are capable of blocking nitrite reductase function in a whole cell assay. We propose that recombinant modified AniA has potential as a vaccine antigen for N. gonorrhoeae.

Role of the Nuclease of Nontypeable Haemophilus influenzae in Dispersal of Organisms from Biofilms

ABSTRACT Nontypeable Haemophilus influenzae (NTHI) forms biofilms in the middle ear during human infection. The biofilm matrix of NTHI contains extracellular DNA. We show that NTHI possesses a potent nuclease, which is a homolog of the thermonuclease of Staphylococcus aureus. Using a biofilm dispersal assay, studies showed a biofilm dispersal pattern in the parent strain, no evidence of dispersal in the nuclease mutant, and a partial return of dispersion in the complemented mutant. Quantitative PCR of mRNA from biofilms from a 24-h continuous flow system demonstrated a significantly increased expression of the nuclease from planktonic organisms compared to those in the biofilm phase of growth (P < 0.042). Microscopic analysis of biofilms grown in vitro showed that in the nuclease mutant the nucleic acid matrix was increased compared to the wild-type and complemented strains. Organisms were typically found in large aggregates, unlike the wild-type and complement biofilms in which the organisms were evenly dispersed throughout the biofilm. At 48 h, the majority of the organisms in the mutant biofilm were dead. The nuclease mutant formed a biofilm in the chinchilla model of otitis media and demonstrated a propensity to also form similar large aggregates of organisms. These studies indicate that NTHI nuclease is involved in biofilm remodeling and organism dispersal.