Bacterial protein domains with a novel Ig‐like fold target human CEACAM receptors

Streptococcus agalactiae, also known as group B Streptococcus (GBS), is the major cause of neonatal sepsis in humans. A critical step to infection is adhesion of bacteria to epithelial surfaces. GBS adhesins have been identified to bind extracellular matrix components and cellular receptors. However, several putative adhesins have no host binding partner characterised. We report here that surface‐expressed β protein of GBS binds to human CEACAM1 and CEACAM5 receptors. A crystal structure of the complex showed that an IgSF domain in β represents a novel Ig‐fold subtype called IgI3, in which unique features allow binding to CEACAM1. Bioinformatic assessment revealed that this newly identified IgI3 fold is not exclusively present in GBS but is predicted to be present in adhesins from other clinically important human pathogens. In agreement with this prediction, we found that CEACAM1 binds to an IgI3 domain found in an adhesin from a different streptococcal species. Overall, our results indicate that the IgI3 fold could provide a broadly applied mechanism for bacteria to target CEACAMs.     

Generation of Human CEACAM1 Transgenic Mice and Binding of Neisseria Opa Protein to Their Neutrophils

Background

Human CEACAM1 is a cell-cell adhesion molecule with multiple functions including insulin clearance in the liver, vasculogenesis in endothelial cells, lumen formation in the mammary gland, and binding of certain human pathogens.

Principal Findings

Three genomic BAC clones containing the human CEACAM1 gene were microinjected into pronuclei of fertilized FVB mouse oocytes. The embryos were implanted in the oviducts of pseudopregnant females and allowed to develop to term. DNA from newborn mice was evaluated by PCR for the presence of the human CEACAM1 gene. Feces of the PCR positive offspring screened for expression of human CEACAM1. Using this assay, one out of five PCR positive lines was positive for human CEACAM1 expression and showed stable transmission to the F1 generation with the expected transmission frequency (0.5) for heterozygotes. Liver, lung, intestine, kidney, mammary gland, and prostate were strongly positive for the dual expression of both murine and human CEACAM1 and mimic that seen in human tissue. Peripheral blood and bone marrow granulocytes stained strongly for human CEACAM1 and bound Neisseria Opa proteins similar to that in human neutrophils.

Conclusion

These transgenic animals may serve as a model for the binding of human pathogens to human CEACAM1.     

Surface displaced alfa-enolase of Lactobacillus plantarum is a fibronectin binding protein

Background  

Lactic acid bacteria of the genus Lactobacillus and Bifidobacterium are one of the most important health promoting groups of the human intestinal microbiota. Their protective role within the gut consists in out competing invading pathogens for ecological niches and metabolic substrates. Among the features necessary to provide health benefits, commensal microorganisms must have the ability to adhere to human intestinal cells and consequently to colonize the gut. Studies on mechanisms mediating adhesion of lactobacilli to human intestinal cells showed that factors involved in the interaction vary mostly among different species and strains, mainly regarding interaction between bacterial adhesins and extracellular matrix or mucus proteins. We have investigated the adhesive properties of Lactobacillus plantarum, a member of the human microbiota of healthy individuals.     

Regulation of CEACAM1 transcription in human breast epithelial cells

Background  

Carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1) is a transmembrane protein with multiple functions in different cell types. CEACAM1 expression is frequently mis-regulated in cancer, with down-regulation reported in several tumors of epithelial origin and de novo expression of CEACAM1 in lung cancer and malignant melanoma. In this report we analyzed the regulation of CEACAM1 expression in three breast cancer cell lines that varied in CEACAM1 expression from none (MCF7) to moderate (MDA-MB-468) to high (MCF10A, comparable to normal breast).     

A novel receptor – ligand pathway for entry of <Emphasis Type="Italic">Francisella tularensis</Emphasis> in monocyte-like THP-1 cells: interaction between surface nucleolin and bacterial elongation factor Tu

Background  

Francisella tularensis, the causative agent of tularemia, is one of the most infectious human bacterial pathogens. It is phagocytosed by immune cells, such as monocytes and macrophages. The precise mechanisms that initiate bacterial uptake have not yet been elucidated. Participation of C3, CR3, class A scavenger receptors and mannose receptor in bacterial uptake have been already reported. However, contribution of an additional, as-yet-unidentified receptor for F. tularensis internalization has been suggested.     

Neisseria meningitidis has two independent modes of recognizing its human receptor CEACAM1

Background

Several human-restricted Gram-negative bacteria exploit carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) for host colonization. For example, Neisseria meningitidis engages these human receptors via outer membrane proteins of the colony opacity-associated (Opa) protein family triggering internalization into non-phagocytic cells.

Principal Findings

We report that a non-opaque strain of N. meningitidis selectively interacts with CEACAM1, but not other CEACAM family members. Using functional assays of bacterial adhesion and internalisation, microscopic analysis, and a panel of CEACAM1 deletion mutants we demonstrate that the engagement of CEACAM1 by non-opaque meningococci occurs in a manner distinct from Opa protein-mediated association. In particular, the amino-terminal domain of CEACAM1 is necessary, but not sufficient for Opa protein-independent binding, which requires multiple extracellular domains of the human receptor in a cellular context. Knock-down of CEACAM1 interferes with binding to lung epithelial cells, whereas chemical or pharmacological disruption of host protein glycosylation does not abrogate CEACAM1 recognition by non-opaque meningococci. The previously characterized meningococcal invasins NadA or Opc do not operate in a CEACAM1-dependent manner.

Conclusions

The results demonstrate a mechanistically distinct, Opa protein-independent interaction between N. meningitidis and human CEACAM1. Our functional investigations suggest the presence of a second CEACAM1-binding invasin on the meningococcal surface that associates with the protein backbone and not the carbohydrate structures of CEACAM1. The redundancy in meningococcal CEACAM1-binding factors further highlights the important role of CEACAM recognition in the biology of this human-adapted pathogen.     

Genotyping of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> clinical isolates in two cities of Turkey: Description of a new family of genotypes that is phylogeographically specific for Asia Minor

Background  

Population-based bacterial genetics using repeated DNA loci is an efficient approach to study the biodiversity and phylogeographical structure of human pathogens, such as Mycobacterium tuberculosis, the agent of tuberculosis. Indeed large genetic diversity databases are available for this pathogen and are regularly updated. No population-based polymorphism data were yet available for M. tuberculosis in Turkey, at the crossroads of Eurasia.     

Bcl10 synergistically links CEACAM3 and TLR‐dependent inflammatory signalling

The neutrophil‐specific innate immune receptor CEACAM3 functions as a decoy to capture Gram‐negative pathogens, such as Neisseria gonorrhoeae, that exploit CEACAM family members to adhere to the epithelium. Bacterial binding to CEACAM3 results in their efficient engulfment and triggers activation of an nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB)‐dependent inflammatory response by human neutrophils. Herein, we report that CEACAM3 cross‐linking is not sufficient for induction of cytokine production and show that the inflammatory response induced by Neisseria gonorrhoeae infection is elicited by an integration of signals from CEACAM3 and toll‐like receptors. Using neutrophils from a human CEACAM‐expressing mouse line (CEABAC), we use a genetic approach to reveal a molecular bifurcation of the CEACAM3‐mediated antimicrobial and inflammatory responses. Ex vivo experiments with CEABAC‐Rac2^?/?, CEABAC‐Bcl10^?/?, and CEABAC‐Malt1^?/? neutrophils indicate that these effectors are not necessary for gonococcal engulfment, yet all 3 effectors contribute to CEACAM3‐mediated cytokine production. Interestingly, although Bcl10 and Malt1 are often inextricably linked, Bcl10 enabled synergy between toll‐like receptor 4 and CEACAM3, whereas Malt1 did not. Together, these findings reveal an integration of the specific innate immune receptor CEACAM3 into the network of more conventional pattern recognition receptors, providing a mechanism by which the innate immune system can unleash its response to a relentless pathogen.     

Phosphatidylinositol 3'-kinase activity is critical for initiating the oxidative burst and bacterial destruction during CEACAM3-mediated phagocytosis

Carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3) is an immunoglobulin-related receptor expressed on human granulocytes. CEACAM3 functions as a single chain phagocytotic receptor recognizing gram-negative bacteria such as Neisseria gonorrhoeae, which possess CEACAM-binding adhesins on their surface. The cytoplasmic domain of CEACAM3 contains an immunoreceptor tyrosine-based activation motif (ITAM)-like sequence that is phosphorylated upon receptor engagement. Here we show that the SH2 domains of the regulatory subunit of phosphatidylinositol 3'-kinase (PI3K) bind to tyrosine residue 230 of CEACAM3 in a phosphorylation-dependent manner. PI3K is rapidly recruited and directly associates with CEACAM3 upon bacterial binding as shown by FRET analysis. Although PI3K activity is not required for efficient uptake of the bacteria by CEACAM3-transfected cells or primary human granulocytes, it is critical for the stimulated production of reactive oxygen species by infected phagocytes and the intracellular degradation of CEACAM-binding bacteria. Together, our results highlight the ability of CEACAM3 to coordinate signaling events that not only mediate bacterial uptake, but also trigger the killing of internalized pathogens.     

Differential disease resistance response in the barley necrotic mutant nec1

Background  

Although ion fluxes are considered to be an integral part of signal transduction during responses to pathogens, only a few ion channels are known to participate in the plant response to infection. CNGC4 is a disease resistance-related cyclic nucleotide-gated ion channel. Arabidopsis thaliana CNGC4 mutants hlm1 and dnd2 display an impaired hypersensitive response (HR), retarded growth, a constitutively active salicylic acid (SA)-mediated pathogenesis-related response and elevated resistance against bacterial pathogens. Barley CNGC4 shares 67% aa identity with AtCNGC4. The barley mutant nec1 comprising of a frame-shift mutation of CNGC4 displays a necrotic phenotype and constitutively over-expresses PR-1, yet it is not known what effect the nec1 mutation has on barley resistance against different types of pathogens.     

Identification of a novel anti-σ<Superscript>E</Superscript> factor in <Emphasis Type="Italic">Neisseria meningitidis</Emphasis>

Background  

Fine tuning expression of genes is a prerequisite for the strictly human pathogen Neisseria meningitidis to survive hostile growth conditions and establish disease. Many bacterial species respond to stress by using alternative σ factors which, in complex with RNA polymerase holoenzyme, recognize specific promoter determinants. σ^E, encoded by rpoE (NMB2144) in meningococci, is known to be essential in mounting responses to environmental challenges in many pathogens. Here we identified genes belonging to the σ^E regulon of meningococci.     

Identification of salivary mucin MUC7 binding proteins from Streptococcus gordonii

Background  

The salivary mucin MUC7 (previously known as MG2) can adhere to various strains of streptococci that are primary colonizers and predominant microorganisms of the oral cavity. Although there is a growing interest in interaction between oral pathogens and salivary mucins, studies reporting the specific binding sites on the bacteria are rather limited. Identification and characterization of the specific interacting proteins on the bacterial cell surface, termed adhesins, are crucial to further understand host-pathogen interactions.     

Adaptation of <Emphasis Type="Italic">Salmonella enterica</Emphasis> Hadar under static magnetic field: effects on outer membrane protein pattern

Background  

Salmonella enterica serovar Hadar (S. Hadar) is a highly prevalent foodborne pathogen and therefore a major cause of human gastroenteritis worldwide. Outer membrane proteins whose production is often regulated by environmental conditions also play important roles in the adaptability of bacterial pathogens to various environments.     

Synergy between type 1 fimbriae expression and C3 opsonisation increases internalisation of E. coli by human tubular epithelial cells

Background  

Bacterial infection of the urinary tract is a common clinical problem with E. coli being the most common urinary pathogen. Bacterial uptake into epithelial cells is increasingly recognised as an important feature of infection. Bacterial virulence factors, especially fimbrial adhesins, have been conclusively shown to promote host cell invasion. Our recent study reported that C3 opsonisation markedly increases the ability of E. coli strain J96 to internalise into human proximal tubular epithelial cells via CD46, a complement regulatory protein expressed on host cell membrane. In this study, we further assessed whether C3-dependent internalisation by human tubular epithelial cells is a general feature of uropathogenic E. coli and investigated features of the bacterial phenotype that may account for any heterogeneity.     

CEACAM6 gene variants in inflammatory bowel disease

Background

The carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) acts as a receptor for adherent-invasive E. coli (AIEC) and its ileal expression is increased in patients with Crohn''s disease (CD). Given its contribution to the pathogenesis of CD, we aimed to investigate the role of genetic variants in the CEACAM6 region in patients with inflammatory bowel diseases (IBD).

Methodology

In this study, a total of 2,683 genomic DNA samples (including DNA from 858 CD patients, 475 patients with ulcerative colitis (UC), and 1,350 healthy, unrelated controls) was analyzed for eight CEACAM6 SNPs (rs10415946, rs1805223 = p.Pro42Pro, rs4803507, rs4803508, rs11548735 = p.Gly239Val, rs7246116 = pHis260His, rs2701, rs10416839). In addition, a detailed haplotype analysis and genotype-phenotype analysis were performed. Overall, our genotype analysis did not reveal any significant association of the investigated CEACAM6 SNPs and haplotypes with CD or UC susceptibility, although certain CEACAM6 SNPs modulated CEACAM6 expression in intestinal epithelial cell lines. Despite its function as receptor of AIEC in ileal CD, we found no association of the CEACAM6 SNPs with ileal or ileocolonic CD. Moreover, there was no evidence of epistasis between the analyzed CEACAM6 variants and the main CD-associated NOD2, IL23R and ATG16L1 variants.

Conclusions

This study represents the first detailed analysis of CEACAM6 variants in IBD patients. Despite its important role in bacterial attachment in ileal CD, we could not demonstrate a role for CEACAM6 variants in IBD susceptibility or regarding an ileal CD phenotype. Further functional studies are required to analyze if these gene variants modulate ileal bacterial attachment.     

The dissemination of C10 cysteine protease genes in <Emphasis Type="Italic">Bacteroides fragilis</Emphasis> by mobile genetic elements

Background  

The C10 family of cysteine proteases includes enzymes that contribute to the virulence of bacterial pathogens, such as SpeB in Streptococcus pyogenes. The presence of homologues of cysteine protease genes in human commensal organisms has not been examined. Bacteroides fragilis is a member of the dominant Bacteroidetes phylum of the human intestinal microbiota, and is a significant opportunistic pathogen.     

The adaptor molecule Nck localizes the WAVE complex to promote actin polymerization during CEACAM3-mediated phagocytosis of bacteria

Background

CEACAM3 is a granulocyte receptor mediating the opsonin-independent recognition and phagocytosis of human-restricted CEACAM-binding bacteria. CEACAM3 function depends on an intracellular immunoreceptor tyrosine-based activation motif (ITAM)-like sequence that is tyrosine phosphorylated by Src family kinases upon receptor engagement. The phosphorylated ITAM-like sequence triggers GTP-loading of Rac by directly associating with the guanine nucleotide exchange factor (GEF) Vav. Rac stimulation in turn is critical for actin cytoskeleton rearrangements that generate lamellipodial protrusions and lead to bacterial uptake.

Principal Findings

In our present study we provide biochemical and microscopic evidence that the adaptor proteins Nck1 and Nck2, but not CrkL, Grb2 or SLP-76, bind to tyrosine phosphorylated CEACAM3. The association is phosphorylation-dependent and requires the Nck SH2 domain. Overexpression of the isolated Nck1 SH2 domain, RNAi-mediated knock-down of Nck1, or genetic deletion of Nck1 and Nck2 interfere with CEACAM3-mediated bacterial internalization and with the formation of lamellipodial protrusions. Nck is constitutively associated with WAVE2 and directs the actin nucleation promoting WAVE complex to tyrosine phosphorylated CEACAM3. In turn, dominant-negative WAVE2 as well as shRNA-mediated knock-down of WAVE2 or the WAVE-complex component Nap1 reduce internalization of bacteria.

Conclusions

Our results provide novel mechanistic insight into CEACAM3-initiated phagocytosis. We suggest that the CEACAM3 ITAM-like sequence is optimized to co-ordinate a minimal set of cellular factors needed to efficiently trigger actin-based lamellipodial protrusions and rapid pathogen engulfment.     

The granulocyte receptor carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3) directly associates with Vav to promote phagocytosis of human pathogens

The human granulocyte-specific receptor carcinoembryonic antigen-related cell adhesion molecule (CEACAM)3 is critically involved in the opsonin-independent recognition of several bacterial pathogens. CEACAM3-mediated phagocytosis depends on the integrity of an ITAM-like sequence within the cytoplasmic domain of CEACAM3 and is characterized by rapid stimulation of the GTPase Rac. By performing a functional screen with CEACAM3-expressing cells, we found that overexpression of a dominant-negative form of the guanine nucleotide exchange factor Vav, but not the dominant-negative versions SWAP70, Dock2, or ELMO1 interfered with CEACAM3-initiated phagocytosis. Moreover, small interfering RNA-mediated silencing of Vav reduced uptake and abrogated the stimulation of Rac in response to bacterial CEACAM3 engagement. In Vav1/Vav2-deficient cells, CEACAM3-mediated internalization was only observed after re-expression of Vav. Vav colocalized with CEACAM3 upon bacterial infection, coimmunoprecipitated in a complex with CEACAM3, and the Vav Src homology 2 domain directly associated with phosphorylated Tyr(230) of CEACAM3. In primary human granulocytes, TAT-mediated transduction of dominant-negative Vav, but not SWAP70, severely impaired the uptake of CEACAM3-binding bacteria. These data support the view that, different from canonical ITAM signaling, the CEACAM3 ITAM-like sequence short-wires bacterial recognition and Rac stimulation via a direct association with Vav to promote rapid phagocytosis and elimination of CEACAM-binding human pathogens.     

Small molecule inhibitors of the Yersinia type III secretion system impair the development of Chlamydia after entry into host cells

Background  

Chlamydiae are obligate intracellular pathogens that possess a type III secretion system to deliver proteins into the host cell during infection. Small molecule inhibitors of type III secretion in Yersinia, termed INPs (Innate Pharmaceuticals AB) were reported to strongly inhibit Chlamydia growth in epithelial cells. In this study we have analyzed the effect of these drugs on bacterial invasiveness.