Airway macrophages display decreased expression of receptors mediating and regulating scavenging in early cystic fibrosis lung disease.

Background: Cystic fibrosis (CF) airway disease is characterized by chronic inflammation, featuring neutrophil influx to the lumen. Airway macrophages (AMs) can promote both inflammation and resolution, and are thus critical to maintaining and restoring homeostasis. CF AM functions, specifically scavenging activity and resolution of inflammation, have been shown to be impaired, yet underlying processes remain unknown. We hypothesized that impaired CF AM function results from an altered expression of receptors that mediate or regulate scavenging, and set out to investigate changes in expression of these markers during the early stages of CF lung disease. Methods: Bronchoalveolar lavage fluid (BALF) was collected from 50 children with CF aged 1, 3 or 5 years. BALF cells were analyzed using flow cytometry. Expression levels of surface markers on AMs were expressed as median fluorescence intensities (MFI) or percentage of AMs positive for these markers. The effect of age and neutrophilic inflammation, among other variables, on marker expression was assessed with a multivariate linear regression model. Results: AM expression of scavenger receptor CD163 decreased with age (p = 0.016) and was negatively correlated with BALF %neutrophils (r = -0.34, p = 0.016). AM expression of immune checkpoint molecule SIRPα also decreased with age (p = 0.0006), but did not correlate with BALF %neutrophils. Percentage of AMs expressing lipid scavenger CD36 was low overall (mean 20.1% ± 16.5) and did not correlate with other factors. Conversely, expression of immune checkpoint PD-1 was observed on the majority of AMs (mean PD-1pos 72.9% ± 11.8), but it, too, was not affected by age or BALF %neutrophils. Compared to matched blood monocytes, AMs had a higher expression of CD16, CD91, and PD-1, and a lower expression of CD163, SIRPα and CD36. Conclusion: In BALF of preschool children with CF, higher age and/or increased neutrophilic inflammation coincided with decreased expression of scavenger receptors on AMs. Expression of scavenging receptors and regulators showed a distinctly different pattern in AMs compared to blood monocytes. These findings suggest AM capacity to counter inflammation and promote homeostasis reduces during initiation of CF airway disease and highlight new avenues of investigation into impaired CF AM function.

Increased CD4(+) T cell co-inhibitory immune receptor CEACAM1 in neonatal sepsis and soluble-CEACAM1 in meningococcal sepsis: a role in sepsis-associated immune suppression?

The co-inhibitory immune receptor carcinoembryonic antigen-related cell-adhesion molecule 1 (CEACAM1) and its self-ligand CEACAM1 can suppress T cell function. Suppression of T cell function in sepsis is well documented. Late-onset neonatal sepsis in VLBW-infants was associated with an increased percentage CEACAM1 positive CD4(+) T-cells. Meningococcal septic shock in children was associated with increased serum soluble CEACAM1. In conclusion our data demonstrate increased surface expression of the co-inhibitory immune receptor CEACAM1 in late-onset neonatal sepsis in VLBW-infants, and increased circulating soluble CEACAM1 in children with meningococcal sepsis. Increased T-cell CEACAM1 expression and increased circulating soluble CEACAM1 may contribute to sepsis-associated immune suppression.

Functional analysis of the superfamily 1 DNA helicases encoded by Mycoplasma pneumoniae and Mycoplasma genitalium.

The DNA recombination and repair machinery of Mycoplasma pneumoniae is composed of a limited set of approximately 11 proteins. Two of these proteins were predicted to be encoded by neighboring open reading frames (ORFs) MPN340 and MPN341. Both ORFs were found to have sequence similarity with genes that encode proteins belonging to the DNA helicase superfamily 1 (SF1). Interestingly, while a homolog of the MPN341 ORF is present in the genome of Mycoplasma genitalium (ORF MG244), MPN340 is an M. pneumoniae-specific ORF that is not found in other mycoplasmas. Moreover, the length of MPN340 (1590 base pairs [bp]) is considerably shorter than that of MPN341 (2148 bp). Examination of the MPN340-encoded amino acid sequence indicated that it may lack a so-called 2B subdomain, which is found in most SF1 DNA helicases. Also, the MPN340-encoded amino acid sequence was found to differ between subtype 1 strain M129 and subtype 2 strain FH at three amino acid positions. Both protein variants, which were termed PcrA(s) M129 and PcrA(s) FH, respectively, as well as the MPN341- and MG244-encoded proteins (PcrA Mpn and PcrA Mge , respectively), were purified, and tested for their ability to interact with DNA. While PcrA Mpn and PcrA Mge were found to bind preferentially to single-stranded DNA, both PcrA(s) M129 and PcrA(s) FH did not demonstrate significant DNA binding. However, all four proteins were found to have divalent cation- and ATP-dependent DNA helicase activity. The proteins displayed highest activity on partially double-stranded DNA substrates carrying 3' single-stranded extensions.

The RuvA homologues from Mycoplasma genitalium and Mycoplasma pneumoniae exhibit unique functional characteristics.

The DNA recombination and repair machineries of Mycoplasma genitalium and Mycoplasma pneumoniae differ considerably from those of gram-positive and gram-negative bacteria. Most notably, M. pneumoniae is unable to express a functional RecU Holliday junction (HJ) resolvase. In addition, the RuvB homologues from both M. pneumoniae and M. genitalium only exhibit DNA helicase activity but not HJ branch migration activity in vitro. To identify a putative role of the RuvA homologues of these mycoplasmas in DNA recombination, both proteins (RuvA(Mpn) and RuvA(Mge), respectively) were studied for their ability to bind DNA and to interact with RuvB and RecU. In spite of a high level of sequence conservation between RuvA(Mpn) and RuvA(Mge) (68.8% identity), substantial differences were found between these proteins in their activities. First, RuvA(Mge) was found to preferentially bind to HJs, whereas RuvA(Mpn) displayed similar affinities for both HJs and single-stranded DNA. Second, while RuvA(Mpn) is able to form two distinct complexes with HJs, RuvA(Mge) only produced a single HJ complex. Third, RuvA(Mge) stimulated the DNA helicase and ATPase activities of RuvB(Mge), whereas RuvA(Mpn) did not augment RuvB activity. Finally, while both RuvA(Mge) and RecU(Mge) efficiently bind to HJs, they did not compete with each other for HJ binding, but formed stable complexes with HJs over a wide protein concentration range. This interaction, however, resulted in inhibition of the HJ resolution activity of RecU(Mge).

Functional characterization of the RuvB homologs from Mycoplasma pneumoniae and Mycoplasma genitalium.

Homologous recombination between repeated DNA elements in the genomes of Mycoplasma species has been hypothesized to be a crucial causal factor in sequence variation of antigenic proteins at the bacterial surface. To investigate this notion, studies were initiated to identify and characterize the proteins that form part of the homologous DNA recombination machinery in Mycoplasma pneumoniae as well as Mycoplasma genitalium. Among the most likely participants of this machinery are homologs of the Holliday junction migration motor protein RuvB. In both M. pneumoniae and M. genitalium, genes have been identified that have the capacity to encode RuvB homologs (MPN536 and MG359, respectively). Here, the characteristics of the MPN536- and MG359-encoded proteins (the RuvB proteins from M. pneumoniae strain FH [RuvB(FH)] and M. genitalium [RuvB(Mge)], respectively) are described. Both RuvB(FH) and RuvB(Mge) were found to have ATPase activity and to bind DNA. In addition, both proteins displayed divalent cation- and ATP-dependent DNA helicase activity on partially double-stranded DNA substrates. The helicase activity of RuvB(Mge), however, was significantly lower than that of RuvB(FH). Interestingly, we found RuvB(FH) to be expressed exclusively by subtype 2 strains of M. pneumoniae. In strains belonging to the other major subtype (subtype 1), a version of the protein is expressed (the RuvB protein from M. pneumoniae strain M129 [RuvB(M129)]) that differs from RuvB(FH) in a single amino acid residue (at position 140). In contrast to RuvB(FH), RuvB(M129) displayed only marginal levels of DNA-unwinding activity. These results demonstrate that M. pneumoniae strains (as well as closely related Mycoplasma spp.) can differ significantly in the function of components of their DNA recombination and repair machinery.

Strain-specific impact of PsaR of Streptococcus pneumoniae on global gene expression and virulence.

Previous studies have indicated that PsaR of Streptococcus pneumoniae is a manganese-dependent regulator, negatively affecting the expression of at least seven genes. Here, we extended these observations by transcriptome and proteome analysis of psaR mutants in strains D39 and TIGR4. The microarray analysis identified three shared PsaR targets: the psa operon, pcpA and prtA. In addition, we found 31 genes to be regulated by PsaR in D39 only, most strikingly a cellobiose-specific phosphotransferase system (PTS) and a putative bacteriocin operon (sp0142-sp0146). In TIGR4, 14 PsaR gene targets were detected, with the rlrA pathogenicity islet being the most pronounced. Proteomics confirmed most of the shared gene targets. To examine the contribution of PsaR to pneumococcal virulence, we compared D39 and TIGR4 wild-type (wt) and psaR mutants in three murine infection models. During colonization, no clear effect was observed of the psaR mutation in either D39 or TIGR4. In the pneumonia model, small but significant differences were observed in the lungs of mice infected with either D39wt or DeltapsaR: D39DeltapsaR had an initial advantage in survival in the lungs. Conversely, TIGR4DeltapsaR-infected mice had significantly lower bacterial loads at 24 h only. Finally, during experimental bacteraemia, D39DeltapsaR-infected mice had significantly lower bacterial loads in the bloodstream than wt-infected mice for the first 24 h of infection. TIGR4DeltapsaR showed attenuation at 36 h only. In conclusion, our results show that PsaR of D39 and TIGR4 has a strain-specific role in global gene expression and in the development of bacteraemia in mice.

Site-specific contributions of glutamine-dependent regulator GlnR and GlnR-regulated genes to virulence of Streptococcus pneumoniae.

The transcriptional regulator GlnR of Streptococcus pneumoniae is involved in the regulation of glutamine and glutamate metabolism, controlling the expression of the glnRA and glnPQ-zwf operons, as well as the gdhA gene. To assess the contribution of the GlnR regulon to virulence, D39 wild-type and mutant strains lacking genes of this regulon were tested in an in vitro adherence assay and murine infection models. All of the mutants, except the DeltaglnR mutant, were attenuated in adherence to human pharyngeal epithelial Detroit 562 cells, suggesting a contribution of these genes to adherence during the colonization of humans. During murine colonization, only the DeltaglnA mutant and the glnP-glnA double mutant (DeltaglnAP) were attenuated, in contrast to DeltaglnP, indicating that the effect is caused by the lack of GlnA expression. In our pneumonia model, only DeltaglnP and DeltaglnAP showed a significantly reduced number of bacteria in the lungs and blood, indicating that GlnP is required for survival in the lungs and possibly for dissemination to the blood. In intravenously infected mice, glnP and glnA were individually dispensable for survival in the blood whereas the DeltaglnAP mutant was avirulent. Finally, transcriptome analysis of the DeltaglnAP mutant showed that many genes involved in amino acid metabolism were upregulated. This signifies the importance of glutamine/glutamate uptake and synthesis for full bacterial fitness and virulence. In conclusion, several genes of the GlnR regulon are required at different sites during pathogenesis, with glnA contributing to colonization and survival in the blood and glnP important for survival in the lungs and, possibly, efficient transition from the lungs to the blood.

Development of lactococcal GEM-based pneumococcal vaccines.

We report the development of a novel protein-based nasal vaccine against Streptococcus pneumoniae, in which three pneumococcal proteins were displayed on the surface of a non-recombinant, killed Lactococcus lactis-derived delivery system, called Gram-positive Enhancer Matrix (GEM). The GEM particles induced the production of the proinflammatory cytokine tumour necrosis factor-alpha (TNF-alpha) by macrophages as well as the maturation of dendritic cells. The pneumococcal proteins IgA1 protease (IgA1p), putative proteinase maturation protein A (PpmA) and streptococcal lipoprotein A (SlrA) were anchored in trans to the surface of the GEM particles after recombinant production of the antigens in L. lactis as hybrids with a lactococcal cell wall binding domain, named Protein Anchor domain (PA). Intranasal immunisation with the SlrA-IgA1p or trivalent vaccine combinations without additional adjuvants showed significant protection against fatal pneumococcal pneumonia in mice. The GEM-based trivalent vaccine is a potential pneumococcal vaccine candidate that is expected to be easy to administer, safe and affordable to produce.

The streptococcal lipoprotein rotamase A (SlrA) is a functional peptidyl-prolyl isomerase involved in pneumococcal colonization.

Streptococcus pneumoniae expresses two surface-exposed lipoproteins, PpmA and SlrA, which share homology with distinct families of peptidyl-prolyl isomerases (PPIases). In this study, we demonstrated for the first time that the lipoprotein cyclophilin, SlrA, can catalyze the cis-trans isomerization of proline containing tetrapeptides and that SlrA contributes to pneumococcal colonization. The substrate specificity of SlrA is typical for prokaryotic and eukaryotic cyclophilins, with Suc-Ala-Ala-Pro-Phe-p-nitroanilide (pNA) being the most rapidly catalyzed substrate. In a mouse pneumonia model the slrA knock-out D39DeltaslrA did not cause significant differences in the survival times of mice compared with the isogenic wild-type strain. In contrast, a detailed analysis of bacterial outgrowth over time in the nasopharynx, airways, lungs, blood, and spleen showed a rapid elimination of slrA mutants from the upper airways but did not reveal significant differences in the lungs, blood, and spleen. These results suggested that SlrA is involved in colonization but does not contribute significantly to invasive pneumococcal disease. In cell culture infection experiments, the absence of SlrA impaired adherence to pneumococcal disease-specific epithelial and endothelial non-professional cell lines. Adherence of the slrA mutant could not be restored by exogenously added SlrA. Strikingly, deficiency in SlrA did not reduce binding activity to host target proteins, but resulted in enhanced uptake by professional phagocytes. In conclusion, SlrA is a functional, cyclophilin-type PPIase and contributes to pneumococcal virulence in the first stage of infection, namely, colonization of the upper airways, most likely by modulating the biological function of important virulence proteins.