Martin Bader

LPS, TLR4 and infectious disease diversity

Innate immune receptors recognize microorganism-specific motifs. One such receptor–ligand complex is formed between the mammalian Toll-like receptor 4 (TLR4)–MD2–CD14 complex and bacterial lipopolysaccharide (LPS). Recent research indicates that there is significant phylogenetic and individual diversity in TLR4-mediated responses. In addition, the diversity of LPS structures and the differential recognition of these structures by TLR4 have been associated with several bacterial diseases. This review will examine the hypothesis that the variability of bacterial ligands such as LPS and their innate immune receptors is an important factor in determining the outcome of infectious disease.

Recognition of Antimicrobial Peptides by a Bacterial Sensor Kinase

PhoQ is a membrane bound sensor kinase important for the pathogenesis of a number of Gram-negative bacterial species. PhoQ and its cognate response regulator PhoP constitute a signal-transduction cascade that controls inducible resistance to host antimicrobial peptides. We show that enzymatic activity of Salmonella typhimurium PhoQ is directly activated by antimicrobial peptides. A highly acidic surface of the PhoQ sensor domain participates in both divalent-cation and antimicrobial-peptide binding as a first step in signal transduction across the bacterial membrane. Identification of PhoQ signaling mutants, binding studies with the PhoQ sensor domain, and structural analysis of this domain can be incorporated into a model in which antimicrobial peptides displace divalent cations from PhoQ metal binding sites to initiate signal transduction. Our findings reveal a molecular mechanism by which bacteria sense small innate immune molecules to initiate a transcriptional program that promotes bacterial virulence.

Regulation of Salmonella typhimurium virulence gene expression by cationic antimicrobial peptides.

Cationic antimicrobial peptides (CAMP) represent a conserved and highly effective component of innate immunity. During infection, the Gram‐negative pathogen Salmonella typhimurium induces different mechanisms of CAMP resistance that promote pathogenesis in animals. This study shows that exposure of S. typhimurium to sublethal concentrations of CAMP activates the PhoP/PhoQ and RpoS virulence regulons, while repressing the transcription of genes required for flagella synthesis and the invasion‐associated type III secretion system. We further demonstrate that growth of S. typhimurium in low doses of the α‐helical peptide C18G induces resistance to CAMP of different structural classes. Inducible resistance depends on the presence of PhoP, indicating that the PhoP/PhoQ system can sense sublethal concentrations of cationic antimicrobial peptides. Growth of S. typhimurium in the presence of CAMP also leads to RpoS‐dependent protection against hydrogen peroxide. Because bacterial resistance to oxidative stress and CAMP are induced during infection of animals, CAMP may be an important signal recognized by bacteria on colonization of animal tissues.

Combined Inhibition of Tumor Necrosis Factor α and Interleukin‐17 As a Therapeutic Opportunity in Rheumatoid Arthritis: Development and Characterization of a Novel Bispecific Antibody

Rheumatoid arthritis therapies that are based on inhibition of a single cytokine, e.g., tumor necrosis factor α (TNFα) or interleukin‐6 (IL‐6), produce clinically meaningful responses in only about half of the treated patients. This study was undertaken to investigate whether combined inhibition of TNFα and IL‐17 has additive or synergistic effects in the suppression of mesenchymal cell activation in vitro and inflammation and tissue destruction in arthritis in vivo.

Comparison of the PhoPQ Regulon in Escherichia coli and Salmonella typhimurium

The PhoPQ two-component system acts as a transcriptional regulator that responds to Mg2+ starvation both in Escherichia coli and Salmonella typhimurium (Garcia et al. 1996; Kato et al. 1999). By monitoring the availability of extracellular Mg2+, this two-component system allows S. typhimurium to sense the transition from an extracellular environment to a subcellular location. Concomitantly with this transition, a set of virulence factors essential for survival in the intracellular environment is activated by the PhoPQ system (Groisman et al. 1989; Miller et al. 1989). Compared to nonpathogenic strains, such as E. coli K12, the PhoPQ regulon in pathogens must contain target genes specifically contributing to the virulence phenotype. To verify this hypothesis, we compared the composition of the PhoPQ regulon between E. coli and S. typhimurium using a combination of expression experiments and motif data. PhoPQ-dependent genes in both organisms were identified from PhoPQ-related microarray experiments. To distinguish between direct and indirect targets, we searched for the presence of the regulatory motif in the promoter region of the identified PhoPQ-dependent genes. This allowed us to reconstruct the direct PhoPQ-dependent regulons in E. coli K12 and S. typhimurium LT2. Comparison of both regulons revealed a very limited overlap of PhoPQ-dependent genes between both organisms. These results suggest that the PhoPQ system has acquired a specialized function during evolution in each of these closely related species that allows adaptation to the specificities of their lifestyles (e.g., pathogenesis in S. typhimurium).

Antimicrobial Peptides Activate the Rcs Regulon through the Outer Membrane Lipoprotein RcsF

Salmonella enterica species are exposed to envelope stresses due to their environmental and infectious lifestyles. Such stresses include amphipathic cationic antimicrobial peptides (CAMPs), and resistance to these peptides is an important property for microbial virulence for animals. Bacterial mechanisms used to sense and respond to CAMP-induced envelope stress include the RcsFCDB phosphorelay, which contributes to survival from polymyxin B exposure. The Rcs phosphorelay includes two inner membrane (IM) proteins, RcsC and RcsD; the response regulator RcsB; the accessory coregulator RcsA; and an outer membrane bound lipoprotein, RcsF. Transcriptional activation of the Rcs regulon occurred within minutes of exposure to CAMP and during the first detectable signs of CAMP-induced membrane disorder. Rcs transcriptional activation by CAMPs required RcsF and preservation of its two internal disulfide linkages. The rerouting of RcsF to the inner membrane or its synthesis as an unanchored periplasmic protein resulted in constitutive activation of the Rcs regulon and RcsCD-dependent phosphorylation. These findings suggest that RcsFCDB activation in response to CAMP-induced membrane disorder is a result of a change in structure or availability of RcsF to the IM signaling constituents of the Rcs phosphorelay.

Escherichia coli Hsp31 functions as a holding chaperone that cooperates with the DnaK-DnaJ-GrpE system in the management of protein misfolding under severe stress conditions

Escherichia coli Hsp31 is a homodimeric protein that exhibits chaperone activity in vitro and is a representative member of a recently recognized family of heat shock proteins (Hsps). To gain insights on Hsp31 cellular function, we deleted the hchA gene from the MC4100 chromosome and combined the resulting null allele with lesions in other cytoplasmic chaperones. Although the hchA mutant only exhibited growth defects when cultivated at 48°C, loss of Hsp31 had a strong deleterious effect on the ability of cells to survive and recover from transient exposure to 50°C, and led to the enhanced aggregation of a subset of host proteins at this temperature. The absence of Hsp31 did not significantly affect the ability of the ClpB‐DnaK‐DnaJ‐GrpE system to clear thermally aggregated proteins at 30°C suggesting that Hsp31 does not possess disaggregase activity. Although it had no effect on the growth of groES30, ΔclpB or ΔibpAB cells at high temperatures, the hchA deletion aggravated the temperature sensitive phenotype of dnaK756 and grpE280 mutants and led to increased aggregation in stressed dnaK756 cells. On the basis of biochemical, structural and genetic data, we propose that Hsp31 acts as a modified holding chaperone that captures early unfolding intermediates under prolonged conditions of severe stress and releases them when cells return to physiological conditions. This additional line of defence would complement the roles of DnaK‐DnaJ‐GrpE, ClpB and IbpB in the management of thermally induced cellular protein misfolding.

The PhoQ histidine kinases of Salmonella and Pseudomonas spp. are structurally and functionally different: evidence that pH and antimicrobial peptide sensing contribute to mammalian pathogenesis

The PhoQ sensor kinase is essential for Salmonella typhimurium virulence for animals, and a homologue exists in the environmental organism and opportunistic pathogen Pseudomonas aeruginosa. S. typhimurium PhoQ (ST‐PhoQ) is repressed by millimolar concentrations of divalent cations and activated by antimicrobial peptides and at acidic pH. ST‐PhoQ has a periplasmic Per‐ARNT‐Sim domain, a fold commonly employed for ligand binding. However, substrate binding is instead accomplished by an acidic patch in the periplasmic domain that interacts with the inner membrane through divalent cation bridges. The DNA sequence encoding this acidic patch is absent from Pseudomonas phoQ (PA‐PhoQ). Here, we demonstrate that PA‐PhoQ binds and is repressed by divalent cations, and can functionally complement a S. typhimurium phoQ‐null mutant. Mutational analysis and NMR spectroscopy of the periplasmic domains of ST‐PhoQ and PA‐PhoQ indicate distinct mechanisms of binding divalent cation. The data are consistent with PA‐PhoQ binding metal in a specific ligand‐binding pocket. PA‐PhoQ was partially activated by acidic pH but not by antimicrobial peptides. S. typhimurium expressing PA‐PhoQ protein were attenuated for virulence in a mouse model, suggesting that the ability of Salmonella to sense host environments via antimicrobial peptides and acidic pH is an important contribution to pathogenesis.

Bacterial Vesicle Formation as a Mechanism of Protein Transfer to Animals

Gram-negative bacterial vesicle formation is a mechanism for specific secretion and transfer of a protein toxin to animals. This discovery should stimulate work on the mechanism of protein sorting into vesicles and the role of vesicles in bacterial pathogenesis.