A scoping review of sexual and reproductive health recommendations in the context of migration to Europe.

In recent years, growing instability and conflict around the world have continued to fuel outward migration, including migration to the EU/EEA. Many migrants hail from countries and regions with a higher burden of STIs-including HIV-and are exposed to enhanced risks of sexual and gender-based violence leading to sexual health issues during their journeys. This scoping review aims to identify existing sexual health recommendations for non-European migrants in the EU/EEA and identify gaps in their implementation. Sexual health recommendations formulated in relation to the migrant population in peer- reviewed journals or by expert consensus, between 2010 and 2021, were included. A keyword search was used to retrieve relevant publications on PubMed, ScienceDirect, the Cochrane Library databases, WHO and ECDC websites. The search strategy employed was charted in a dedicated Prisma Chart. Overall, 180 publications were retrieved. Based on the abstract and after eliminating duplicates, 33 publications were included for full-text reading. The references of these publications were screened. In total, 19 publications met the inclusion criteria. Evidence-based sexual health recommendations target only newly arrived migrants and migrant children (at arrival and during stay in the country). Screening practices are mostly informed by the country of origin and related prevalence, which remains a limitation; challenges faced during migration should also be considered. Implementation and compliance with these recommendations remain uncertain, as sexual health is not funded and addressed in a uniform manner across Europe.

Exposure to Secreted Bacterial Factors Promotes HIV-1 Replication in CD4+ T Cells.

Microbial translocation is associated with systemic immune activation in HIV-1 disease. Circulating T cells can encounter microbial products in the bloodstream and lymph nodes, where viral replication takes place. The mechanisms by which bacteria contribute to HIV-associated pathogenesis are not completely deciphered. Here, we examined how bacteria may impact T cell function and viral replication. We established cocultures between a panel of live bacteria and uninfected or HIV-1-infected activated peripheral blood CD4-positive (CD4+) T cells. We show that some bacteria, such as Escherichia coli and Acinetobacter baumannii, sustain lymphocyte activation and enhance HIV-1 replication. Bacteria secrete soluble factors that upregulate CD25 and ICAM-1 cell surface levels and activate NF-κB nuclear translocation. Our data also demonstrate that CD25 polarizes at the virological synapse, suggesting a previously unappreciated role of CD25 during viral replication. These findings highlight how interactions between bacterial factors and T cells may promote T cell activation and HIV-1 replication. IMPORTANCE People living with HIV suffer from chronic immune activation despite effective antiretroviral therapy. Early after infection, HIV-1 actively replicates in the gut, causing the breakage of the intestinal epithelial barrier and microbial translocation. Microbial translocation and chronic immune activation have been proven linked; however, gaps in our knowledge on how bacteria contribute to the development of HIV-related diseases remain. Whether T cells in the peripheral blood react to bacterial products and how this affects viral replication are unknown. We show that some bacteria enriched in people living with HIV activate T cells and favor HIV-1's spread. Bacteria release soluble factors that cause the overexpression of cellular molecules related to their activation state. T cells overexpressing these molecules also replicate HIV-1 more efficiently. These results help us learn more about how HIV-1, T cells, and bacteria interact with each other, as well as the mechanisms behind chronic immune activation.

Azithromycin analogue CSY0073 attenuates lung inflammation induced by LPS challenge.

BACKGROUND AND PURPOSE: Azithromycin is a macrolide antibiotic with anti-inflammatory and immunomodulating effects. Long-term azithromycin therapy in patients with chronic lung diseases such as cystic fibrosis has been associated with increased antimicrobial resistance, emergence of hypermutable strains, ototoxicity and cardiac toxicity. The aim of this study was to assess the anti-inflammatory effects of the non-antibiotic azithromycin derivative CSY0073. EXPERIMENTAL APPROACH: We compared the effects of CSY0073 with those of azithromycin in experiments on bacterial cultures, Pseudomonas aeruginosa biofilm, lung cells and mice challenged intranasally with P. aeruginosa LPS. KEY RESULTS: In contrast to azithromycin, CSY0073 did not inhibit the growth of P. aeruginosa, Staphylococcus aureus or Haemophilus influenzae and had no effect on an established P. aeruginosa biofilm. Bronchoalveolar lavage (BAL) fluids and lung homogenates collected after the LPS challenge in mice showed that CSY0073 and azithromycin (200 mg·kg(-1), i.p.) decreased neutrophil counts at 24 h and TNF-α, CXCL1 and CXCL2 levels in the BAL fluid after 3 h and IL-6, CXCL2 and IL-1ß levels in the lung after 3 h compared with the vehicle. However, only azithromycin reduced IL-1ß levels in the lung 24 h post LPS challenge. CSY0073 and azithromycin similarly diminished the production of pro-inflammatory cytokines by macrophages, but not lung epithelial cells, exposed to P. aeruginosa LPS. CONCLUSIONS AND IMPLICATIONS: Unlike azithromycin, CSY0073 had no antibacterial effects but it did have a similar anti-inflammatory profile to that of azithromycin. Hence, CSY0073 may have potential as a long-term treatment for patients with chronic lung diseases.

Dynamic modulation of fimbrial extension and FimH-mannose binding force on live bacteria under pH changes: a molecular atomic force microscopy analysis.

Mechanical and conformational properties of type 1 fimbriae were evaluated on live bacterial cells by Single Molecule Force Spectroscopy (SMFS) and Dynamic Force Spectroscopy (DFS) in buffered solutions whose pH varied from 3 to 9. We evidenced that both fimbrial extension and fimbrial binding force to mannosylated-surface are modulated with changing the externally applied shear force and the solution pH. In particular, intertwined FimA-FimA and FimH-mannose interactions lead to a 5 to 25-fold decrease of the fimbrial unwinding for pulling rates larger than 10 µm/s and for pH values outside the range 5 to 7. In this pH range, the FimH-mannose binding force is maximal with a magnitude of -150-200 pN and the fimbriae extension reaches 8 µm. The enhancement of the FimH-mannose binding force at neutral pH, as evidenced from molecular AFM analyses, strongly correlates with an optimum in yeast agglutination detected at pH 5 to 7. The results reported in this work suggest that "catch bond effect" was negligible over the range of pulling rates tested, and both FimA-FimA and FimH-mannose interactions under given pH and external shear force conditions modify the ability of the bacteria to efficiently colonize host surfaces.

Escherichia coli biofilms.

Escherichia coli is a predominant species among facultative anaerobic bacteria of the gastrointestinal tract. Both its frequent community lifestyle and the availability of a wide array of genetic tools contributed to establish E. coli as a relevant model organism for the study of surface colonization. Several key factors, including different extracellular appendages, are implicated in E. coli surface colonization and their expression and activity are finely regulated, both in space and time, to ensure productive events leading to mature biofilm formation. This chapter will present known molecular mechanisms underlying biofilm development in both commensal and pathogenic E. coli.

Haemophore-mediated signalling in Serratia marcescens: a new mode of regulation for an extra cytoplasmic function (ECF) sigma factor involved in haem acquisition.

Bacterial extra cytoplasmic function (ECF) sigma factors control a wide range of cell envelope activities including iron and haem uptake systems. Sigma activity is usually inhibited by membrane-bound antisigma. An extra cytoplasmic signal modulates sigma-antisigma interactions and thereby leads to the transcription of the target operon. Sigma and antisigma genes generally belong to one autoregulated operon. However, ECF sigma and antisigma genes involved in iron acquisition, also called iron starvation ECF, are non-autoregulated exceptions to this rule. We fully reconstituted the has signalling cascade of Serratia marcescens in Escherichia coli. Binding of the haem-loaded haemophore to the outer membrane receptor, HasR, inactivates the antisigma HasS, turning on HasI and thereby allowing has operon transcription. Deletion of the HasR N-terminal extension, present in all characterized outer membrane receptors endowed with signal transduction capacity, abolished the inducing activity but not the transport activity. Induction required the TonB-ExbB-ExbD complex. HasI, like the other iron starvation sigma, is iron repressed but not autoregulated. We found an entirely new regulation for the antisigma hasS gene, the transcription of which is HasI dependent. We suggest that the has system is both activated and repressed by the availability of external haem. When there is enough haem, the HasS antisigma activity is turned off and HasI induces the transcription of hasS. This leads to the storage of inactive HasS molecules which become active when HasR is not occupied by holo-haemophore ligand molecules: as soon as there is a haem shortage transcription is turned off. Positive autoregulation of ECF sigma and antisigma genes is usually considered as a mechanism for amplifying a perceived signal. However, our findings suggest, on the contrary, that antisigma regulation allows fine-tuning to the external signal. The biological significance of ECF sigma and antisigma autoregulation may need to be reconsidered.

Characterization of HasB, a Serratia marcescens TonB-like protein specifically involved in the haemophore-dependent haem acquisition system.

In Gram-negative bacteria, the TonB-ExbB-ExbD inner membrane multiprotein complex is required for active transport of diverse molecules through the outer membrane. We present evidence that Serratia marcescens, like several other Gram-negative bacteria, has two TonB proteins: the previously characterized TonBSM, and also HasB, a newly identified component of the has operon that encodes a haemophore-dependent haem acquisition system. This system involves a soluble extracellular protein (the HasA haemophore) that acquires free or haemoprotein-bound haem and presents it to a specific outer membrane haemophore receptor (HasR). TonBSM and HasB are significantly similar and can replace each other for haem acquisition. However, TonBSM, but not HasB, mediates iron acquisition from iron sources other than haem and haemoproteins, showing that HasB and TonBSM only display partial redundancy. The reconstitution in Escherichia coli of the S. marcescens Has system demonstrated that haem uptake is dependent on the E. coli ExbB, ExbD and TonB proteins and that HasB is non-functional in E. coli. Nevertheless, a mutation in the HasB transmembrane anchor domain allows it to replace TonBEC for haem acquisition. As the change affects a domain involved in specific TonBEC-ExbBEC interactions, HasB may be unable to interact with ExbBEC, and the HasB mutation may allow this interaction. In E. coli, the HasB mutant protein was functional for haem uptake but could not complement the other TonBEC-dependent functions, such as iron siderophore acquisition, and phage DNA and colicin uptake. Our findings support the emerging hypothesis that TonB homologues are widespread in bacteria, where they may have specific functions in receptor-ligand uptake systems.

Identification and characterization of the hemophore-dependent heme acquisition system of Yersinia pestis.

Yersinia pestis possesses a heme-protein acquisition system (Hmu) that allows it to utilize heme and heme-protein complexes as the sole sources of iron. Analysis of the Y. pestis CO92 genomic sequence revealed a second heme-protein acquisition gene cluster that shares homology with the hemophore-dependent heme acquisition system (Has system) of Serratia marcescens. This locus consisted of the hasR(yp) receptor gene, the hasA(yp) hemophore gene, and genes encoding components of the HasA(yp) dedicated ABC transporter factor (hasDE(yp)), as well as a tonB homologue (hasB(yp)). By using a reconstituted secretion system in Escherichia coli, we showed that HasA(yp) is a secreted heme-binding protein and that expression of HasA(yp) is iron regulated in E. coli. The use of a transcriptional reporter fusion showed that the hasRADEB promoter is Fur regulated and has increased activity at 37 degrees C. Hemoglobin utilization via the Has(yp) system was studied with both E. coli and Y. pestis, for which has and has hmu mutant strains were used. No contribution of the Has system to heme utilization was observed in either E. coli or Y. pestis under the conditions we tested. Previously it was shown that a deletion of the Hmu system had no effect on the virulence of Y. pestis in a mouse model of bubonic plague. An Hmu(-) Has(-) double mutant also retained full virulence in this model of infection. This report constitutes the first attempt to investigate the contribution of the hemophore-dependent heme acquisition system in bacterial pathogenicity.

Natural conjugative plasmids induce bacterial biofilm development.

Horizontal gene transfer is a principal source of evolution leading to change in the ecological character of bacterial species. Bacterial conjugation, which promotes the horizontal transfer of genetic material between donor and recipient cells by physical contact, is a phenomenon of fundamental evolutionary consequence. Although conjugation has been studied primarily in liquid, most natural bacterial populations are found associated with environmental surfaces in complex multispecies communities called biofilms. Biofilms are ideally suited to the exchange of genetic material of various origins, and it has been shown that bacterial conjugation occurs within biofilms. Here I investigate the direct contribution of conjugative plasmids themselves to the capacity of the bacterial host to form a biofilm. Natural conjugative plasmids expressed factors that induced planktonic bacteria to form or enter biofilm communities, which favour the infectious transfer of the plasmid. This general connection between conjugation and biofilms suggests that medically relevant plasmid-bearing strains are more likely to form a biofilm. This may influence both the chances of biofilm-related infection risks and of conjugational spread of virulence factors.

A rapid method for efficient gene replacement in the filamentous fungus Aspergillus nidulans.

The construction of mutant fungal strains is often limited by the poor efficiency of homologous recombination in these organisms. Higher recombination efficiencies can be obtained by increasing the length of homologous DNA flanking the transformation marker, although this is a tedious process when standard molecular biology techniques are used for the construction of gene replacement cassettes. Here, we present a two-step technology which takes advantage of an Escherichia coli strain expressing the phage lambda Red(gam, bet, exo) functions and involves (i) the construction in this strain of a recombinant cosmid by in vivo recombination between a cosmid carrying a genomic region of interest and a PCR-generated transformation marker flanked by 50 bp regions of homology with the target DNA and (ii) genetic exchange in the fungus itself between the chromosomal locus and the circular or linearized recombinant cosmid. This strategy enables the rapid establishment of mutant strains carrying gene knock-outs with efficiencies >50%. It should also be appropriate for the construction of fungal strains with gene fusions or promoter replacements.

Cell division in Escherichia coli: role of FtsL domains in septal localization, function, and oligomerization.

In Escherichia coli, nine essential cell division proteins are known to localize to the division septum. FtsL is a 13-kDa bitopic membrane protein with a short cytoplasmic N-terminal domain, a membrane-spanning segment, and a periplasmic domain that has a repeated heptad motif characteristic of leucine zippers. Here, we identify the requirements for FtsL septal localization and function. We used green fluorescent protein fusions to FtsL proteins where domains of FtsL had been exchanged with analogous domains from either its Haemophilus influenzae homologue or the unrelated MalF protein to show that both the membrane-spanning segment and the periplasmic domain of FtsL are required for localization to the division site. Mutagenesis of the periplasmic heptad repeat motif severely impaired both localization and function as well as the ability of FtsL to drive the formation of sodium dodecyl sulfate-resistant multimers in vitro. These results are consistent with the predicted propensity of the FtsL periplasmic domain to adopt a coiled-coiled structure. This coiled-coil motif is conserved in all gram-negative and gram-positive FtsL homologues identified so far. Our data suggest that most of the FtsL molecule is a helical coiled coil involved in FtsL multimerization.

Localization of FtsL to the Escherichia coli septal ring.

In Escherichia coli, nine gene products are known to be essential for assembly of the division septum. One of these, FtsL, is a bitopic membrane protein whose precise function is not understood. Here we use fluorescence microscopy to study the subcellular localization of FtsL, both in a wild-type strain and in a merodiploid strain that expresses a GFP-FtsL fusion protein. We show that FtsL localizes to the cell septum where it forms a ring analogous to the cytoplasmic FtsZ ring. FtsL localization is dependent upon the function of FtsZ, FtsA and FtsQ, but not FtsI. In a reverse approach, we use fusions of green fluorescent protein (GFP) to FtsZ, FtsA and ZipA to show that these proteins localize to the division site in an FtsL-independent fashion. We propose that FtsL is a relatively late recruit to the ring structure that mediates septation.

Localization of FtsI (PBP3) to the septal ring requires its membrane anchor, the Z ring, FtsA, FtsQ, and FtsL.

Assembly of the division septum in bacteria is mediated by several proteins that localize to the division site. One of these, FtsI (also called penicillin-binding protein 3) of Escherichia coli, consists of a short cytoplasmic domain, a single membrane-spanning segment, and a large periplasmic domain that encodes a transpeptidase activity involved in synthesis of septal peptidoglycan. We have constructed a merodiploid strain with a wild-type copy of ftsI at the normal chromosomal locus and a genetic fusion of ftsI to the green fluorescent protein (gfp) at the lambda attachment site. gfp-ftsI was expressed at physiologically appropriate levels under control of a regulatable promoter. Consistent with previous results based on immunofluorescence microscopy GFP-FtsI localized to the division site during the later stages of cell growth and throughout septation. Localization of GFP-FtsI to the cell pole(s) was not observed unless the protein was overproduced about 10-fold. Membrane anchor alterations shown previously to impair division but not membrane insertion or transpeptidase activity were found to interfere with localization of GFP-FtsI to the division site. In contrast, GFP-FtsI localized well in the presence of beta-lactam antibiotics that inhibit the transpeptidase activity of FtsI. Septal localization depended upon every other division protein tested (FtsZ, FtsA, FtsQ, and FtsL). We conclude that FtsI is a late recruit to the division site, and that its localization depends on an intact membrane anchor.

Septal localization of FtsQ, an essential cell division protein in Escherichia coli.

Septation in Escherichia coli requires several gene products. One of these, FtsQ, is a simple bitopic membrane protein with a short cytoplasmic N terminus, a membrane-spanning segment, and a periplasmic domain. We have constructed a merodiploid strain that expresses both FtsQ and the fusion protein green fluorescent protein (GFP)-FtsQ from single-copy chromosomal genes. The gfp-ftsQ gene complements a null mutation in ftsQ. Fluorescence microscopy revealed that GFP-FtsQ localizes to the division site. Replacing the cytoplasmic and transmembrane domains of FtsQ with alternative membrane anchors did not prevent the localization of the GFP fusion protein, while replacing the periplasmic domain did, suggesting that the periplasmic domain is necessary and sufficient for septal targeting. GFP-FtsQ localization to the septum depended on the cell division proteins FtsZ and FtsA, which are cytoplasmic, but not on FtsL and FtsI, which are bitopic membrane proteins with comparatively large periplasmic domains. In addition, the septal localization of ZipA apparently did not require functional FtsQ. Our results indicate that FtsQ is an intermediate recruit to the division site.

A new type of hemophore-dependent heme acquisition system of Serratia marcescens reconstituted in Escherichia coli.

The utilization by Serratia marcescens of heme bound to hemoglobin requires HasA, an extracellular heme-binding protein. This unique heme acquisition system was studied in an Escherichia coli hemA mutant that was a heme auxotroph. We identified a 92-kDa iron-regulated S. marcescens outer membrane protein, HasR, which alone enabled the E. coli hemA mutant to grow on heme or hemoglobin as a porphyrin source. The concomitant secretion of HasA by the HasR-producing hemA mutant greatly facilitates the acquisition of heme from hemoglobin. This is the first report of a synergy between an outer membrane protein and an extracellular heme-binding protein, HasA, acting as a heme carrier, which we termed a hemophore.

Protein secretion by Gram-negative bacterial ABC exporters--a review.

One of the strategies used by Gram-negative bacteria to secrete proteins across the two membranes which delimit the cells is sec-independent and dedicated to proteins lacking an N-terminal signal peptide. Most of these proteins display a C-terminal secretion signal located in the last 60 amino acids (aa). Using one Erwinia chrysanthemi protease, PrtG, secreted by such a pathway it was shown that the smallest C-terminal sequence allowing efficient secretion contains the last 29 aa of PrtG and that low but significant secretion can be promoted by the last 15 aa of PrtG. Moreover, the extreme C-terminal motif, consisting of a negatively charged aa followed by several hydrophobic aa must be exposed and is conserved amongst many proteins following this pathway. This secretion system depends on ABC protein-mediated exporters, which consist of three cell envelope proteins: two inner membrane proteins, an ATPase (the ABC protein), a membrane fusion protein (MFP) and an outer membrane polypeptide. These Gram-negative bacterial protein exporters are dedicated to the secretion of one or several closely related proteins belonging to the toxin, protease and lipase families. The genes encoding the three secretion proteins and the exoproteins are usually all linked, consistent with the specificity of the systems. Er. chrysanthemi metalloproteases B and C and Serratia marcescens hemoprotein HasA are secreted by such homologous pathways and interact with the ABC protein. Interaction between the ABC protein and its substrate has also been evidenced by studies on protease and HasA hybrid transporters obtained by combining components from each system. Association between hemoprotein HasA and the three exporter secretion proteins was demonstrated by affinity chromatography on hemin agarose on which the substrate remained bound with the three secretion proteins. The three components' association was ordered and substrate binding was required for the formation of this multiprotein complex.

Protein secretion by gram-negative bacterial ABC exporters.

One of the strategies used by Gram-negative bacteria to secrete proteins across the two membranes which delimit the cells, is sec independent and dedicated to proteins lacking an N-terminal signal peptide. It depends on ABC protein-mediated exporters, which consist of three cell envelope proteins: two inner membrane proteins: an ATPase (the ABC protein), a membrane fusion protein (MFP) and an outer membrane polypeptide. Erwinia chrysanthemi metalloproteinases B and C, and Serratia marcescens hemoprotein HasA are secreted by such homologous pathways and interact with the ABC protein. Interaction between the ABC protein and its substrate has also been evidenced by studies on proteinase and HasA hybrid transporters obtained by combining components from each system. Association between hemoprotein HasA and the three exporter/secretion proteins was demonstrated by affinity chromatography on hemin agarose on which the substrate remained bound with the three secretion proteins. The three component association was ordered and substrate binding was required for the formation of this multiprotein complex.

Spectroscopic studies of the C-terminal secretion signal of the Serratia marcescens haem acquisition protein (HasA) in various membrane-mimetic environments.

The structure of a peptide comprising the last 56 C-terminal residues of the Serratia marcescens haem acquisition protein (HasA) secreted by an ATP-binding cassette exporter was examined by 1H-NMR, circular dichroic and fluorescence spectroscopies. The peptide, which contains the secretion signal of HasA, is efficiently secreted by the HasA transporter. It is largely unstructured and flexible in aqueous buffer solution, but its helical content increases upon addition of trifluoroethanol, detergents and lipids. By circular dichroism, a stable helical conformation is observed between 20% and 70% (by vol.) trifluoroethanol. The 1H-NMR spectrum was analysed at these two trifluoroethanol concentrations; residues 7-15, 21-30 and 40-50 were shown to form relatively stable helices. In the presence of neutral detergent, alpha-helix is induced to a similar extent upon micelle formation; in this case, fluorescence data indicate that at least the N-terminus of the peptide interacts with the micelle. In the presence of negatively charged detergent, alpha-helix is induced before micelle formation and the N-terminus of the peptide seems not to be involved in this interaction. In the presence of negatively charged liposomes, the peptide interacts with the vesicle, again inducing a helical conformation. However, the helical content remains lower than upon addition of trifluoroethanol or neutral micelles. These results are compared to those previously obtained with the secretion signal of one of the Erwinia chrysanthemi metalloproteases which are transported efficiently by the HasA transporter. Both signals exhibit similar conformational features, despite their low sequence similarity.

Iron acquisition from heme and hemoglobin by a Serratia marcescens extracellular protein.

Several pathogenic bacteria are able to use heme and hemoproteins as iron sources independent of siderophore production by mechanisms involving outer membrane heme-binding proteins and heme transport systems. Here we show that Serratia marcescens has such a property and we identify an extracellular heme-binding protein, HasA (for heme acquisition system), allowing the release of heme from hemoglobin. This protein is secreted by S. marcescens under conditions of iron depletion and is essential for heme acquisition.

Secretion of the Serratia marcescens HasA protein by an ABC transporter.

We previously identified a Serratia marcescens extracellular protein, HasA, able to bind heme and required for iron acquisition from heme and hemoglobin by the bacterium. This novel type of extracellular protein does not have a signal peptide and does not show sequence similarities to other proteins. HasA secretion was reconstituted in Escherichia coli, and we show here that like many proteins lacking a signal peptide, HasA has a C-terminal targeting sequence and is secreted by a specific ATP binding cassette (ABC) transporter consisting of three proteins, one inner membrane protein with a conserved ATP binding domain, called the ABC; a second inner membrane protein; and a third, outer membrane component. Since the three S. marcescens components of the HasA transporter have not yet been identified, the reconstituted HasA secretion system is a hybrid. It consists of the two S. marcescens inner membrane-specific components, HasD and HasE, associated with an outer membrane component coming from another bacterial ABC transporter, such as the E. coli TolC protein, the outer membrane component of the hemolysin transporter, or the Erwinia chrysanthemi PrtF protein, the outer membrane component of the protease transporter. This hybrid transporter was first shown to allow the secretion of the S. marcescens metalloprotease and the E. chrysanthemi metalloproteases B and C. On account of that, the two S. marcescens components HasD and HasE were previously named PrtDSM and PrtESM, respectively. However, HasA is secreted neither by the PrtD-PrtE-PrtF transporter (the genuine E. chrysanthemi protease transporter) nor by the HlyB-HlhD-TolC transporter (the hemolysin transporter). Moreover, HasA, coexpressed in the same cell, strongly inhibits the secretion of proteases B and C by their own transporter, indicating that the E. chrysanthemi transporter recognizes HasA. Since PrtF could replace TolC in the constitution of the HasA transporter, this indicates that the secretion block does not take place at the level of the outer membrane component but rather at an earlier step of interaction between HasA and the inner membrane components.