Stapling of Peptides Potentiates the Antibiotic Treatment of Acinetobacter baumannii In Vivo

The rising incidence of multidrug resistance in Gram-negative bacteria underlines the urgency for novel treatment options. One promising new approach is the synergistic combination of antibiotics with antimicrobial peptides. However, the use of such peptides is not straightforward; they are often sensitive to proteolytic degradation, which greatly limits their clinical potential. One approach to increase stability is to apply a hydrocarbon staple to the antimicrobial peptide, thereby fixing them in an α-helical conformation, which renders them less exposed to proteolytic activity. In this work we applied several different hydrocarbon staples to two previously described peptides shown to act on the outer membrane, L6 and L8, and tested their activity in a zebrafish embryo infection model using a clinical isolate of Acinetobacter baumannii as a pathogen. We show that the introduction of such a hydrocarbon staple to the peptide L8 improves its in vivo potentiating activity on antibiotic treatment, without increasing its in vivo antimicrobial activity, toxicity or hemolytic activity

Two-Partner Secretion Systems of Neisseria meningitidis Associated with Invasive Clonal Complexes▿ †

The two-partner secretion (TPS) pathway is widespread among gram-negative bacteria and facilitates the secretion of very large and often virulence-related proteins. TPS systems consist of a secreted TpsA protein and a TpsB protein involved in TpsA transport across the outer membrane. Sequenced Neisseria meningitidis genomes contain up to five TpsA- and two TpsB-encoding genes. Here, we investigated the distribution of TPS-related open reading frames in a collection of disease isolates. Three distinct TPS systems were identified among meningococci. System 1 was ubiquitous, while systems 2 and 3 were significantly more prevalent among isolates of hyperinvasive clonal complexes than among isolates of poorly invasive clonal complexes. In laboratory cultures, systems 1 and 2 were expressed. However, several sera from patients recovering from disseminated meningococcal disease recognized the TpsAs of systems 2 and 3, indicating the expression of these systems during infection. Furthermore, we showed that the major secreted TpsAs of systems 1 and 2 depend on their cognate TpsBs for transport across the outer membrane and that the system 1 TpsAs undergo processing. Together, our data indicate that TPS systems may contribute to the virulence of N. meningitidis

On display:Autotransporter secretion and application

The classical monomeric autotransporters are ubiquitously used by Gram-negative bacteria to export virulence and colonization factors to their cell surface or into their surroundings. They are expressed as monomeric proteins that pass the inner and outer membrane in two consecutive steps facilitated by the Sec translocon and the Bam complex, respectively. In this mini-review we discuss how autotransporters translocate their secreted functional domains across the outer membrane. We highlight the interactions with the Bam complex and discuss how specific features of the recently solved structure of Bam lead to a mechanistic model for autotransporter secretion. Furthermore, the autotransporter secretion pathway is the system of choice for surface display of heterologous proteins for biotechnical and biomedical purposes. We summarize recent advances in the application of autotransporters with a focus on outer membrane vesicle vaccine development and discuss its limitations in secreting more complex heterologous proteins. Finally, we present an exciting new technology to circumvent secretion limitations by ligating heterologous proteins of interest to autotransporters that are displayed on the cell surface

Stress-based high-throughput screening assays to identify inhibitors of cell envelope biogenesis

The structural integrity of the Gram-negative cell envelope is guarded by several stress responses, such as the σE, Cpx and Rcs systems. Here, we report on assays that monitor these responses in E. coli upon addition of antibacterial compounds. Interestingly, compromised peptidoglycan synthesis, outer membrane biogenesis and LPS integrity predominantly activated the Rcs response, which we developed into a robust HTS (high-throughput screening) assay that is suited for phenotypic compound screening. Furthermore, by interrogating all three cell envelope stress reporters, and a reporter for the cytosolic heat-shock response as control, we found that inhibitors of specific envelope targets induce stress reporter profiles that are distinct in quality, amplitude and kinetics. Finally, we show that by using a host strain with a more permeable outer membrane, large-scaffold antibiotics can also be identified by the reporter assays. Together, the data suggest that stress profiling is a useful first filter for HTS aimed at inhibitors of cell envelope processes

NalP-Mediated Proteolytic Release of Lactoferrin-Binding Protein B from the Meningococcal Cell Surface ▿

Bacteria have developed several mechanisms for iron uptake during colonization of mammalian hosts, where the availability of free iron is limiting for growth. Neisseria meningitidis expresses under iron-limiting conditions a receptor complex consisting of the lactoferrin-binding proteins A (LbpA) and LbpB to acquire iron from lactoferrin, which is abundantly present on the mucosal surfaces of the human nasopharynx. LbpA is an integral outer membrane-embedded iron transporter, whereas LbpB is a cell surface-exposed lipoprotein. In this study, we demonstrate that LbpB is also released into the culture medium. We identified NalP, an autotransporter known to be involved in the processing of other autotransporters, as the protease responsible for LbpB release. This release of LbpB reduced the complement-mediated killing of the bacteria when incubated with an LbpB-specific bactericidal antiserum. Since antibodies directed against LbpB are found in convalescent-patient sera, the release of an immunogenic protein as LbpB may represent a novel means for N. meningitidis to escape the human immune response

Structure of the translocator domain of a bacterial autotransporter

Autotransporters are virulence-related proteins of Gram-negative bacteria that are secreted via an outer-membrane-based C-terminal extension, the translocator domain. This domain supposedly is sufficient for the transport of the N-terminal passenger domain across the outer membrane. We present here the crystal structure of the in vitro-folded translocator domain of the autotransporter NalP from Neisseria meningitidis, which reveals a 12-stranded β-barrel with a hydrophilic pore of 10 × 12.5 Å that is filled by an N-terminal α-helix. The domain has pore activity in vivo and in vitro. Our data are consistent with the model of passenger-domain transport through the hydrophilic channel within the β-barrel, and inconsistent with a model for transport through a central channel formed by an oligomer of translocator domains. However, the dimensions of the pore imply translocation of the secreted domain in an unfolded form. An alternative model, possibly covering the transport of folded domains, is that passenger-domain transport involves the Omp85 complex, the machinery required for membrane insertion of outer-membrane proteins, on which autotransporters are dependent

A Functional Carbohydrate Degrading Enzyme Potentially Acquired by Horizontal Gene Transfer in the Genome of the Soil Invertebrate Folsomia candida

Horizontal gene transfer (HGT) is defined as the acquisition by an organism of hereditary material from a phylogenetically unrelated organism. This process is mostly observed among bacteria and archaea, and considered less likely between microbes and multicellular eukaryotes. However, recent studies provide compelling evidence of the evolutionary importance of HGT in eukaryotes, driving functional innovation. Here, we study an HGT event in Folsomia candida (Collembola, Hexapoda) of a carbohydrate-active enzyme homologous to glycosyl hydrase group 43 (GH43). The gene encodes an N-terminal signal peptide, targeting the product for excretion, which suggests that it contributes to the diversity of digestive capacities of the detritivore host. The predicted α-L-arabinofuranosidase shows high similarity to genes in two other Collembola, an insect and a tardigrade. The gene was cloned and expressed in Escherichia coli using a cell-free protein expression system. The expressed protein showed activity against p-nitrophenyl-α-L-arabinofuranoside. Our work provides evidence for functional activity of an HGT gene in a soil-living detritivore, most likely from a bacterial donor, with genuine eukaryotic properties, such as a signal peptide. Co-evolution of metazoan GH43 genes with the Panarthropoda phylogeny suggests the HGT event took place early in the evolution of this ecdysozoan lineage

Overexpression of the Bam Complex Improves the Production of Chlamydia trachomatis MOMP in the E. coli Outer Membrane

A licensed Chlamydia trachomatis (Ct) vaccine is not yet available. Recombinant Chlamydia trachomatis major outer membrane protein (Ct-MOMP), the most abundant constituent of the chlamydial outer membrane complex, is considered the most attractive candidate for subunit-based vaccine formulations. Unfortunately, Ct-MOMP is difficult to express in its native structure in the E. coli outer membrane (OM). Here, by co-expression of the Bam complex, we improved the expression and localization of recombinant Ct-MOMP in the E. coli OM. Under these conditions, recombinant Ct-MOMP appeared to assemble into a β-barrel conformation and express domains at the cell surface indicative of correct folding. The data indicate that limited availability of the Bam complex can be a bottleneck for the production of heterologous OM vaccine antigens, information that is also relevant for strategies aimed at producing recombinant OMV-based vaccines

Characterization of Adherence of Nontypeable Haemophilus influenzae to Human Epithelial Cells

The adherence of 58 nontypeable Haemophilus influenzae isolates obtained from patients with otitis media or chronic obstructive pulmonary disease (COPD) and obtained from the throats of healthy individuals to Chang and NCI-H292 epithelial cells was compared. Otitis media isolates, but not COPD isolates, adhered significantly more to both cell lines than did throat isolates. Since high-molecular-weight (HMW) proteins are major adhesins of nontypeable H. influenzae, the isolates were screened for HMW protein expression by Western blotting with two polyclonal sera and PCR with hmw-specific primers. Twenty-three of the 32 adhering isolates (72%) and only 1 of the 26 nonadherent strains were HMW protein or hmw gene positive. Among the 32 isolates adhering to either cell line, 5 different adherence patterns were distinguished based on the inhibiting effect of dextran sulfate. Using H. influenzae strain 12 expressing two well-defined HMW proteins (HMW1 and HMW2) and its isogenic mutants as a reference, we observed HMW1-like adherence to both cell lines for 16 of the 32 adherent isolates. Four others showed HMW2-like adherence to NCI-H292. Of the three other patterns of adherence, one probably also involved HMW protein. Screening of the isolates with six HMW-specific monoclonal antibodies in a whole-cell enzyme-linked immunosorbent assay showed that the HMW proteins of COPD isolates and carrier isolates were more distinct from the HMW proteins from H. influenzae strain 12 than those from otitis media isolates. Characterization of the HMW protein of a COPD isolate by adherence and DNA sequence analysis showed that despite large sequence diversity in the hmwA gene, probably resulting in the antigenic differences, the HMW protein mediated the HMW2-like adherence of this strain