Binding of human factor H to outer membrane protein P5 of non‐typeable Haemophilus influenzae contributes to complement resistance

Non‐typeable Haemophilus influenzae is an opportunistic pathogen of the human upper respiratory tract and is often found to cause inflammatory diseases that include sinusitis, otitis media and exacerbations of chronic obstructive pulmonary disease. To persist in the inflammatory milieu during infection, non‐typeable H. influenzae must resist the antimicrobial activity of the human complement system. Here, we used Tn‐seq to identify genes important for resistance to complement‐mediated killing. This screen identified outer membrane protein P5 in evasion of the alternative pathway of complement activation. Outer membrane protein P5 was shown to bind human complement regulatory protein factor H directly, thereby, preventing complement factor C3 deposition on the surface of the bacterium. Furthermore, we show that amino acid variation within surface‐exposed regions within outer membrane P5 affected the level of factor H binding between individual strains.     

Impact of sequence diversity in the Moraxella catarrhalis UspA2/UspA2H head domain on vitronectin binding and antigenic variation

The nasopharyngeal pathogen Moraxella catarrhalis recruits vitronectin to subvert complement-mediated killing. Ubiquitous surface protein (UspA) 2 and its hybrid form UspA2H bind vitronectin at the highly diverse N-terminal head domain. Here we characterized the sequence diversity of the head domain in multiple M. catarrhalis clinical isolates (n = 51) with focus on binding of vitronectin. The head domain of the uspA2 genes from 40 isolates were clustered according to an N-terminal sequence motif of UspA2 (NTER2), i.e., NTER2A (55% of uspA2 variants), NTER2B (32.5%), NTER2C (5%), and finally a group without an NTER2 (7.5%). Isolates harbouring the uspA2H gene (n = 11) contained N-terminal GGG repeats. Vitronectin binding to isolates having UspA2 did not correlate to variation in the NTER2 motifs but occurred in UspA2H containing 6 or ≥11 of GGG repeats. Analyses of recombinant UspA2/UspA2H head domains of multiple variants showed UspA2-dependent binding to the C-terminal of vitronectin. Furthermore, polyclonal anti-UspA2 antibodies revealed that the head domain of the majority of Moraxella UspA2/2H was antigenically unrelated, whereas the full length molecules were recognized. In conclusion, the head domains of UspA2/2H have extensive sequence polymorphism without losing vitronectin-binding capacity promoting a general evasion of the host immune system.     

Blood treatment of Lyme borreliae demonstrates the mechanism of CspZ‐mediated complement evasion to promote systemic infection in vertebrate hosts

Lyme disease, caused by the spirochete Borrelia burgdorferi, is the most common vector‐borne disease in the United States and Europe. The spirochetes are transmitted from mammalian and avian reservoir hosts to humans via ticks. Following tick bites, spirochetes colonize the host skin and then disseminate haematogenously to various organs, a process that requires this pathogen to evade host complement, an innate immune defence system. CspZ, a spirochete surface protein, facilitates resistance to complement‐mediated killing in vitro by binding to the complement regulator, factor H (FH). Low expression levels of CspZ in spirochetes cultivated in vitro or during initiation of infection in vivo have been a major hurdle in delineating the role of this protein in pathogenesis. Here, we show that treatment of B. burgdorferi with human blood induces CspZ production and enhances resistance to complement. By contrast, a cspZ‐deficient mutant and a strain that expressed an FH‐nonbinding CspZ variant were impaired in their ability to cause bacteraemia and colonize tissues of mice or quail; virulence of these mutants was however restored in complement C3‐deficient mice. These novel findings suggest that FH binding to CspZ facilitates B. burgdorferi complement evasion in vivo and promotes systemic infection in vertebrate hosts.     

Expanding the paradigm for the outer membrane: Acinetobacter baumannii in the absence of endotoxin

Asymmetry in the outer membrane has long defined the cell envelope of Gram‐negative bacteria. This asymmetry, with lipopolysaccharide (LPS) or lipooligosaccharide (LOS) exclusively in the outer leaflet of the membrane, establishes an impermeable barrier that protects the cell from a number of stressors in the environment. Work done over the past 5 years has shown that Acinetobacter baumannii has the remarkable capability to survive with inactivated production of lipid A biosynthesis and the absence of LOS in its outer membrane. The implications of LOS‐deficient A. baumannii are far‐reaching – from impacts on cell envelope biogenesis and maintenance, bacterial physiology, antibiotic resistance and virulence. This review examines recent work that has contributed to our understanding of LOS‐deficiency and compares it to studies done on Neisseria meningitidis and Moraxella catarrhalis; the two other organisms with this capability.     

Genes essential for phototrophic growth by a purple alphaproteobacterium

Tn‐seq was used to identify genes essential for phototrophic growth by the purple bacterium Rhodopseudomonas palustris. About 167 genes required for anaerobic growth on acetate in light were identified, 35 of which are annotated as photosynthesis genes. The essentiality of many of these genes by analysing the phenotypes of independently generated mutants that had altered pigmentation was verified. Three genes were identified, two possibly involved in biogenesis of the membrane‐bound photosynthetic apparatus and one for phosphatidylcholine biosynthesis, that were not known to be essential for phototrophic growth. Site‐directed mutagenesis was used to show that the NADH:quinone oxidoreductase complex I_E was essential for phototrophic growth under strictly anaerobic conditions and appeared to play a role in reverse electron transport to generate NADH. A homologous NADH:quinone oxidoreductase complex I_A likely operates in the opposite direction to oxidize NADH. The operation of the two enzymes in opposition would allow R. palustris to maintain redox balance. As a complement to the genetic data, proteomics experiments were carried out in which it was found that 408 proteins were present in significantly higher amounts in cells grown anaerobically in light compared with aerobically. Among these were proteins encoded by subset of the phototrophic growth‐essential genes.     

Genome‐wide identification of tolerance mechanisms toward p‐coumaric acid in Pseudomonas putida

The soil bacterium Pseudomonas putida KT2440 has gained increasing biotechnological interest due to its ability to tolerate different types of stress. Here, the tolerance of P. putida KT2440 toward eleven toxic chemical compounds was investigated. P. putida was found to be significantly more tolerant toward three of the eleven compounds when compared to Escherichia coli. Increased tolerance was for example found toward p‐coumaric acid, an interesting precursor for polymerization with a significant industrial relevance. The tolerance mechanism was therefore investigated using the genome‐wide approach, Tn‐seq. Libraries containing a large number of miniTn5‐Km transposon insertion mutants were grown in the presence and absence of p‐coumaric acid, and the enrichment or depletion of mutants was quantified by high‐throughput sequencing. Several genes, including the ABC transporter Ttg2ABC and the cytochrome c maturation system (ccm), were identified to play an important role in the tolerance toward p‐coumaric acid of this bacterium. Most of the identified genes were involved in membrane stability, suggesting that tolerance toward p‐coumaric acid is related to transport and membrane integrity.

     

Complementation of transposition of tnpA mutants of Tn3, Tn21, Tn501, and Tn1721

The transposons Tn21, Tn501, and Tn1721 are related to Tn3. Transposition-deficient mutants (tnpA) of these elements were used to test for complementation of transpostion. Transposition of tnpA mutants of Tn501 and Tn1721 was restored by the presence in trans of Tn21, Tn501, and Tn1721, but transposition of a tnpA mutant of Tn21 was restored in trans only by Tn21 itself. Tn3 did not complement transposition of Tn21, Tn501, or Tn1721, and these elements did not complement transposition of Tn3.     

Bactericidal Monoclonal Antibody Against <Emphasis Type="Italic">Moraxella catarrhalis</Emphasis> Lipooligosaccharide Cross-Reacts with <Emphasis Type="Italic">Haemophilus Spp.</Emphasis>

Monoclonal antibodies (MAbs) against lipooligosaccharide (LOS) determinants after immunization of BALB/c mice with heat inactivated Moraxella catarrhalis serotype A were generated. MAb 219A9 was specific for a common epitope of A, B, and C M. catarrhalis serotypes in ELISA and immunofluorescent test (IFT). In both tests it also cross-reacted with whole bacteria and LPS antigens isolated from non-typeable H. influenzae and H. parainfluenzae strains. IgM antibody clone 219A9 possessed a strong bactericidal effect against the three serotypes in the presence of complement. Our results demonstrate that antibodies directed to a single LOS epitope common for A, B, and C serotype could be highly protective. This suggests that the common determinants are very promising in the development of LOS-based vaccine against M. catarrhalis. The cross-reactions of MAb 219A9 with Haemophilus spp. also show that immunization could result in immune response to epitopes conserved in other important respiratory pathogens.     

MoNSTR‐seq,a restriction site‐associated DNA sequencing technique to characterize Agrobacterium‐mediated transfer‐DNA insertions in Phomopsis longicolla

Phomopsis longicolla (Hobbs) causes Phomopsis seed decay and stem lesions in soybean (Glycine max). In this study, a novel, high‐throughput adaptation of RAD‐seq termed MoNSTR‐seq (Mutation analysis via Next‐generation DNA Sequencing of T‐DNA Regions) was developed to determine the genomic location of T‐DNA insertions in P. longicolla mutants. Insertional mutants were created via Agrobacterium tumefaciens‐mediated transformation, and one mutant, strain PL343, was further investigated due to impaired stem lesion formation. Mutation analysis via Next‐generation DNA Sequencing of T‐DNA Regions, in which DNA libraries are created with two distinct restriction enzymes and customized adapters to simultaneously enrich both T‐DNA insertion borders, was developed to characterize the genomic lesion in strain PL343. MoNSTR‐seq successfully identified a T‐DNA insertion in the predicted promoter region of a gene encoding a cellobiose dehydrogenase (CDH1), and the position of the T‐DNA insertion in strain PL343 was confirmed by Sanger sequencing. Thus, MoNSTR‐seq represents an effective tool for molecular genetics in P. longicolla, and is readily adaptable for use in diverse fungal species.

Significance and Impact of the Study

This study describes MoNSTR‐seq (Mutation analysis via Next‐generation DNA Sequencing of T‐DNA Regions), an adaptation of restriction site‐associated DNA sequencing (RAD‐seq) to identify the position of transfer‐DNA (T‐DNA) insertions in the genome of Phomopsis longicolla, an important pathogen of soybean. The technique enables high‐throughput characterization of mutants generated via Agrobacterium tumefaciens‐mediated transformation (ATMT), thus accelerating gene discovery via forward genetics. This technique represents a significant advancement over existing approaches to characterize T‐DNA insertions in fungal genomes. With minor modifications, this technique could be easily adapted to taxonomically diverse fungal pathogens and additional mutagenesis cassettes.     

Genes for phaseolotoxin synthesis are located on the chromosome ofPseudomonas syringae pv.phaseolicola

Transposon mutagenesis was used to locate the genes for phaseolotoxin biosynthesis inPseudomonas syringae pathovar.phaseolicola. Mutants unable to produce toxin were obtained that carried Tn5 on different chromosomal restriction fragments. None of the Tn5-induced nontoxigenic mutants carried the transposon in plasmid DNA. The insertion of Tn5 intotox DNA was confirmed by site-directed mutagenesis. The results reported here suggest the involvement of at least five chromosomal genes in phaseolotoxin biosynthesis. All of the toxinminus mutants retained both full pathogenicity on beans and resistance to the toxin.     

BGA66 and BGA71 facilitate complement resistance of Borrelia bavariensis by inhibiting assembly of the membrane attack complex

Borrelia (B.) bavariensis exhibits a marked tropism for nervous tissues and frequently causes neurological manifestations in humans. The molecular mechanism by which B. bavariensis overcomes innate immunity, in particular, complement remains elusive. In contrast to other serum‐resistant spirochetes, none of the B. bavariensis isolates investigated bound complement regulators of the alternative (AP) and classical pathway (CP) or proteolytically inactivated complement components. Focusing on outer surface proteins BGA66 and BGA71, we demonstrated that both molecules either inhibit AP, CP and terminal pathway (TP) activation, or block activation of the CP and TP respectively. Both molecules bind complement components C7, C8 and C9, and thereby prevent assembly of the terminal complement complex. This inhibitory activity was confirmed by the introduction of the BGA66 and BGA71 encoding genes into a serum‐sensitive B. garinii strain. Transformed spirochetes producing either BGA66 or BGA71 overcome complement‐mediated killing, thus indicating that both proteins independently facilitate serum resistance of B. bavariensis. The generation of C‐terminally truncated proteins as well as a chimeric BGA71 protein lead to the localization of the complement‐interacting binding site within the N‐terminus. Collectively, our data reveal a novel immune evasion strategy of B. bavariensis that is directed against the activation of the TP.     

Anomalies of the anaerobic tricarboxylic acid cycle in Shewanella oneidensis revealed by Tn‐seq

The availability of increasingly inexpensive sequencing combined with an ever‐expanding molecular biology toolbox has transported classical bacterial genetics into the 21st century. Whole genome genetic fitness analysis using transposon mutagenesis combined with next‐generation high‐throughput sequencing (Tn‐seq) promises to revolutionize systems level analysis of microbial metabolism. Tn‐seq measures the frequency of actual members of a heterogeneous mutant pool undergoing purifying selection to determine the contribution of every non‐essential gene in the genome to the fitness of an organism under a given condition. Here we use Tn‐seq to assess gene function in the Gram negative γ‐proteobacterium Shewanella oneidensis strain MR‐1. In addition to being a model environmental organism, there is considerable interest in using S. oneidensis as a platform organism for bioremediation and biotechnology, necessitating a complete understanding of the metabolic pathways that may be utilized. Our analysis reveals unique aspects of S. oneidensis metabolism overlooked by over 30 years of classical genetic and systems level analysis. We report the utilization of an alternative citrate synthase and describe a dynamic branching of the S. oneidensis anaerobic tricarboxylic acid cycle, unreported in any other organism, which may be a widespread strategy for microbes adept at dissipating reducing equivalents via anaerobic respiration.     

Vitronectin binds to the head region of Moraxella catarrhalis ubiquitous surface protein A2 and confers complement‐inhibitory activity

The serum resistance of the common respiratory pathogen Moraxella catarrhalis is mainly dependent on ubiquitous surface proteins (Usp) A1 and A2 that interact with complement factor 3 (C3) and complement inhibitor C4b binding protein (C4BP) preventing the alternative and classical pathways of the complement system respectively. UspA2 also has the capacity to attract vitronectin that in turn binds C9 and hereby inhibits membrane attack complex (MAC) formation. We found UspA2 as a major vitronectin binding protein and hence the UspA2/vitronectin interaction was studied in detail. The affinity constant (K_D) for vitronectin binding to UspA2 was 2.3 × 10^?8 M, and the N‐terminal region encompassing residues UspA2 30–170 bound vitronectin with a K_D of 7.9 × 10^?8 M. Electron microscopy verified that the active binding domain (UspA2^30–177) was located at the head region of UspA2. Experiments with recombinantly expressed vitronectin also revealed that UspA2^30–177 bound to the C‐terminal region of vitronectin residues 312–396. Finally, when human serum was pre‐incubated with UspA2, bacteria showed significantly less serum resistance. Our study directly reveals the binding mode between the N‐terminal domain of UspA2 and the C‐terminal part of vitronectin and thus sheds light upon the mechanism of M. catarrhalis‐dependent serum resistance.     

The Bordetella pertussis Bps polysaccharide enhances lung colonization by conferring protection from complement‐mediated killing

Bordetella pertussis is a human‐restricted Gram‐negative bacterial pathogen that causes whooping cough or pertussis. Pertussis is the leading vaccine preventable disease that is resurging in the USA and other parts of the developed world. There is an incomplete understanding of the mechanisms by which B. pertussis evades killing and clearance by the complement system, a first line of host innate immune defence. The present study examined the role of the Bps polysaccharide to resist complement activity in vitro and in the mouse respiratory tract. The isogenic bps mutant strain containing a large non‐polar in‐frame deletion of the bpsA–D locus was more sensitive to serum and complement mediated killing than the WT strain. As determined by Western blotting, flow cytometry and electron microscopic studies, the heightened sensitivity of the mutant strain was due to enhanced deposition of complement proteins and the formation of membrane attack complex, the end‐product of complement activation. Bps was sufficient to confer complement resistance as evidenced by a Bps‐expressing Escherichia coli being protected by serum killing. Additionally, Western blotting and flow cytometry assays revealed that Bps inhibited the deposition of complement proteins independent of other B. pertussis factors. The bps mutant strain colonized the lungs of complement‐deficient mice at higher levels than that observed in C57Bl/6 mice. These results reveal a previously unknown interaction between Bps and the complement system in controlling B. pertussis colonization of the respiratory tract. These findings also make Bps a potential target for the prevention and therapy of whooping cough.