Targeting intracellular, multi-drug resistant Staphylococcus aureus with guanidinium polymers by elucidating the structure-activity relationship

Intracellular persistence of bacteria represents a clinical challenge as bacteria can thrive in an environment protected from antibiotics and immune responses. Novel targeting strategies are critical in tackling antibiotic resistant infections. Synthetic antimicrobial peptides (SAMPs) are interesting candidates as they exhibit a very high antimicrobial activity. We first compared the activity of a library of ammonium and guanidinium polymers with different sequences (statistical, tetrablock and diblock) synthesized by RAFT polymerization against methicillin-resistant S. aureus (MRSA) and methicillin-sensitive strains (MSSA). As the guanidinium SAMPs were the most potent, they were used to treat intracellular S. aureus in keratinocytes. The diblock structure was the most active, reducing the amount of intracellular MSSA and MRSA by two-fold. We present here a potential treatment for intracellular, multi-drug resistant bacteria, using a simple and scalable strategy

The type VII secretion system protects Staphylococcus aureus against antimicrobial host fatty acids

The Staphylococcus aureus type VII secretion system (T7SS) exports several proteins that are pivotal for bacterial virulence. The mechanisms underlying T7SS-mediated staphylococcal survival during infection nevertheless remain unclear. Here we report that S. aureus lacking T7SS components are more susceptible to host-derived antimicrobial fatty acids. Unsaturated fatty acids such as linoleic acid (LA) elicited an increased inhibition of S. aureus mutants lacking T7SS effectors EsxC, EsxA and EsxB, or the membrane-bound ATPase EssC, compared to the wild-type (WT). T7SS mutants generated in different S. aureus strain backgrounds also displayed an increased sensitivity to LA. Analysis of bacterial membrane lipid profiles revealed that the esxC mutant was less able to incorporate LA into its membrane phospholipids. Although the ability to bind labelled LA did not differ between the WT and mutant strains, LA induced more cell membrane damage in the T7SS mutants compared to the WT. Furthermore, proteomic analyses of WT and mutant cell fractions revealed that, in addition to compromising membranes, T7SS defects induce oxidative stress and hamper their response to LA challenge. Thus, our findings indicate that T7SS contribute to maintaining S. aureus membrane integrity and homeostasis when bacteria encounter antimicrobial fatty acids

Identification and characterization of the novel CEACAM-binding adhesin of Haemophilus influenzae

Haemophilus influenzae (Hinf), a mostly commensal inhabitant of human respiratory airways, is the causative agent of local infections such as middle ear infections, sinusitis, bronchitis, conjunctivitis and pneumonia, but can also cause life-threatening disseminating diseases, including meningitis and septicemia. The success of Hinf as commensal or opportunistic pathogen relies on the ability of the bacterium to use a combination of adhesive surface proteins, the so-called adhesins, which intimately bind to structures on the human mucosal tissue in order to overcome mechanical clearance and escape immune recognition. In that respect, Hinf targets carcinoembryonic antigen-related cell adhesion molecules (CEACAMs), which are glycoproteins of the immunoglobulin superfamily and which are present on the apical side of nasopharyngeal epithelial cells. The first chapter of this work presents in detail the numerous advantages for the bacteria inherent in their interaction with epithelial CEACAMs (e.g.: strong attachment to the mucosal surface, internalization within epithelial cells and therefore protection against immune response).The interaction between Hinf and CEACAMs was postulated to be mediated by the outer membrane protein (OMP) P5, one of the major OMPs of Hinf. Therefore, we set out to characterize the molecular requirements of CEACAM-binding by OMP P5. However, (i) the ability of P5-deficient Hinf to strongly interact with CEACAMs; (ii) the similarity of P5 sequences from non-CEACAM-binding and CEACAM-binding Hinf strains, and (iii) the failure of OMP P5 to bind CEACAMs when heterologously expressed in Escherichia coli questioned the role of OMP P5 as the Hinf CEACAM-binding adhesin. A screen of a panel of Hinf mutants with defined deletions in their outer membrane proteins revealed that the depletion of OMP P1 totally abrogated the interaction of Hinf with CEACAMs. Furthermore, non-CEACAM-binding E. coli gained CEACAM-binding properties upon ectopic expression of Hinf OMP P1. Thus, OMP P1 is necessary and sufficient for CEACAM-targeting.Following the identification of OMP P1 as the bona fide CEACAM-binding adhesin of Hinf, we sought to identify the domain(s) of OMP P1 involved in CEACAM-recognition. Therefore, we took advantage of the high similarity between OMP P1 and its non-CEACAM-binding homolog FadL in E. coli. The solved crystal structure of FadL helped to model OMP P1, which is predicted to build a 14-stranded β-barrel. Interestingly, chimeras resulting from the fusion of OMP P1 with FadL fragments combined with OMP P1 mutants deprived of specific extracellular loops clearly underlined the involvement of the most prominent surface-exposed OMP P1 loops (L1, L3, L4 and L7) in CEACAM recognition. The flexibility of these loops appears important for the proper binding to CEACAM. In contrast to Hinf OMP P1, P1 homologs of other members of the Pasteurellaceae family were unable to recognize CEACAMs. Moreover, Hinf OMP P1 bound exclusively to human CEACAMs, but not to other distantly related mammalian CEACAMs.FadL, the OMP P1 homolog in E. coli, mediates the transport of long-chain fatty acids (LCFAs) across the bacterial outer membrane. The high structural homology shared by FadL and OMP P1 raised the question whether OMP P1 is involved in fatty acid transport and/or is regulated by fatty acids. Though the addition of fatty acids to the Hinf medium was able to potently stimulate bacterial growth, the role of OMP P1 in LCFA transport requires further investigations. However, it was obvious that LCFAs supplementations upregulated OMP P1 expression in Hinf. Furthermore, Hinf strains treated with LCFAs were able to better interact with recombinant CEACAMs and were more invasive in CEACAM-expressing epithelial cells, compared to untreated bacteria.In sum, this study not only identifies a novel CEACAM-binding adhesin in a prominent human pathogen, but also provides insights into a new CEACAM-binding interface built by 4 flexible loops of a bacterial outer membrane protein.publishe

Signaling by epithelial members of the CEACAM Family - mucosal docking sites for pathogenic bacteria

Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) comprise a group of immunoglobulin-related vertebrate glycoproteins. Several family members, including CEACAM1, CEA, and CEACAM6, are found on epithelial tissues throughout the human body. As they modulate diverse cellular functions, their signaling capacity is in the focus of current research. In this review we will summarize the knowledge about common signaling processes initiated by epithelial CEACAMs and suggest a model of signal transduction by CEACAM family members lacking significant cytoplasmic domains. As pathogenic and non-pathogenic bacteria exploit these receptors during mucosal colonization, we try to highlight the connection between CEACAMs, microbes, and cellular responses. Special emphasis in this context is placed on the functional interplay between CEACAMs and integrins that influences matrix adhesion of epithelial cells. The cooperation between these two receptor families provides an intriguing example of the fine tuning of cellular responses and their manipulation by specialized microorganisms

Uropathogenic E. coli Exploit CEA to Promote Colonization of the Urogenital Tract Mucosa

Attachment to the host mucosa is a key step in bacterial pathogenesis. On the apical surface of epithelial cells, members of the human carcinoembryonic antigen (CEA) family are abundant glycoproteins involved in cell-cell adhesion and modulation of cell signaling. Interestingly, several gram-negative bacterial pathogens target these receptors by specialized adhesins. The prototype of a CEACAM-binding pathogen, Neisseria gonorrhoeae, utilizes colony opacity associated (Opa) proteins to engage CEA, as well as the CEA-related cell adhesion molecules CEACAM1 and CEACAM6 on human epithelial cells. By heterologous expression of neisserial Opa proteins in non-pathogenic E. coli we find that the Opa protein-CEA interaction is sufficient to alter gene expression, to increase integrin activity and to promote matrix adhesion of infected cervical carcinoma cells and immortalized vaginal epithelial cells in vitro. These CEA-triggered events translate in suppression of exfoliation and improved colonization of the urogenital tract by Opa protein-expressing E. coli in CEA-transgenic compared to wildtype mice. Interestingly, uropathogenic E. coli expressing an unrelated CEACAM-binding protein of the Afa/Dr adhesin family recapitulate the in vitro and in vivo phenotype. In contrast, an isogenic strain lacking the CEACAM-binding adhesin shows reduced colonization and does not suppress epithelial exfoliation. These results demonstrate that engagement of human CEACAMs by distinct bacterial adhesins is sufficient to blunt exfoliation and to promote host infection. Our findings provide novel insight into mucosal colonization by a common UPEC pathotype and help to explain why human CEACAMs are a preferred epithelial target structure for diverse gram-negative bacteria to establish a foothold on the human mucosa.publishe

Uropathogenic <i>E</i>. <i>coli</i> expressing the AfaE-III adhesin selectively bind to the amino terminal domain of human CEACAM family members.

<p><i>(A)</i> The indicated soluble CEACAM-GFP fusion proteins (human CEACAM1-NT, CEA-NT, and CEACAM8-NT) were produced in 293 cells and the resulting cell culture supernatants were incubated with <i>E</i>. <i>coli</i> AfaE-III or with the AfaE-III-deficient strain (<i>E</i>. <i>coli</i> ΔAfaE-III). After washing, bacteria-associated CEACAM-GFP fusion proteins were detected by Western blotting with a monoclonal anti-GFP antibody (Pull-down; upper two panels). To verify the presence of equal amounts of the CEACAM-GFP fusion proteins, cell culture supernatants were analyzed by Western blotting with anti-GFP antibody (Supe; lowest panel). Shown is a representative experiment out of three independent biological replicates. (<i>B</i>) Cell culture supernatants containing the soluble N-terminal domains of murine CEACAM1 (mCEA1), bovine CEACAM1a (bCEA1a), bovine CEACAM1b (bCEA1b), canine CEACAM1 (cCEA1), human CEACAM1 (hCEA1) as GFP-fusion proteins, or GFP alone (GFP) were incubated with <i>E</i>. <i>coli</i> AfaE-III. After washing, bacteria-associated CEACAM1 was detected as in (A) (Pull-down; upper panel). The presence of CEACAM-GFP fusion proteins was analyzed as in (A) (Supe; lower panel). Shown is a representative experiment out of three independent biological replicates. <i>(C)</i> ME-180 cells were infected or not with the indicated bacteria for 2 h, fixed and co-stained with antibodies against endogenous CEACAMs (clone D14HD11; red) and rabbit anti-<i>E</i>. <i>coli</i> (green). Arrowheads indicate bacteria bound to clustered CEA. Pictures are representative for four independent biological replicates.</p

<i>E</i>. <i>coli</i> AfaE-III suppresses epithelial exfoliation in CEAtg mice.

<p><i>(A)</i> Whole mount urogenital tracts of uninfected wild-type or CEAtg female mice were fixed and processed for scanning electron microscopy (SEM). Pictures show the luminal surface of the upper vaginal and cervical regions and are representative for five animals each treatment group. <i>(B)</i> Wild-type or CEAtg female mice were infected with <i>E</i>. <i>coli</i> AfaE-III or <i>E</i>. <i>coli</i> ΔAfaE-III for 24 h and the genital tracts processed as in (A). SEM pictures (at two different magnifications, as indicated by the scale bars) show the luminal surface of the upper vaginal and cervical regions. Whereas massive epithelial exfoliation is evident in infected wildtype mice and in CEAtg mice infected with <i>E</i>. <i>coli</i> ΔAfaE-III, a strongly reduced detachment of epithelial cells is observed in CEAtg mice infected with CEACAM-binding <i>E</i>. <i>coli</i> AfaE-III. Adherent bacteria can be observed at higher magnifications (arrows). Pictures are representative for five animals each treatment group. <i>(C)</i> Quantification of exfoliating epithelial cells from samples in (B). Bars represent mean ± S.D. of exfoliating cells from at least n = 26 areas (~0.075 mm²) derived from at least five animals each treatment group. Results were compared by Mann-Whitney U-test and highly significant differences (p<0.001) are indicated by ***.</p

The interaction of <i>E</i>. <i>coli</i> Opa_CEA with CEA facilitates mucosal colonization and leads to CD105 expression in epithelial cells.

<p><i>(A)</i> Wild-type (●) or CEAtg (▼) female mice were infected with the indicated bacterial strains and 24 h later, bacteria were re-isolated. Each data point in the graph reflects the number of bacteria re-isolated from an individual animal (n = 10; except for CEAtg animals infected with <i>E</i>. <i>coli</i> Opa_CEA, where n = 15). Data were compiled from four independent experiments. The median for each experimental group of animals is indicated by a line; groups were compared by Mann-Whitney U-test and highly significant differences (p<0.001) are indicated by ***. <i>(B)</i> Individual re-isolated bacterial colonies from the genital tract of CEAtg mice infected with either <i>E</i>. <i>coli</i> or <i>E</i>. <i>coli</i> Opa_CEA were plated on LB-ampicillin agar plates (post infection). Five isolates for each strain were analysed by Western blotting with an antibody against Opa protein. As a control, lysates of the <i>E</i>. <i>coli</i> or <i>E</i>. <i>coli</i> Opa_CEA used for infection (input) were also analysed. <i>(C)</i> Genital tracts from CEAtg mice infected for 24 hours with <i>E</i>. <i>coli</i> or <i>E</i>. <i>coli</i> Opa_CEA were excised, and cryosections were co-stained with antibodies against <i>E</i>. <i>coli</i> (green) and against CEA (red). Cell nuclei were stained with Hoechst dye (blue). Arrowheads indicate host-associated <i>E</i>. <i>coli</i>. Pictures are representative for three independent biological replicates. <i>(D)</i> Cryosections as in (C) were co-stained with antibodies against <i>E</i>. <i>coli</i> (green) and a rat monoclonal antibody against murine CD105 (red). Cell nuclei were visualized by Hoechst (blue). CD105 expression on the mucosal surface of CEAtg mice infected with <i>E</i>. <i>coli</i> Opa_CEA is highlighted by small arrows. Pictures are representative for three independent biological replicates.</p