From microbial gene essentiality to novel antimicrobial drug targets.

BACKGROUND: Bacterial respiratory tract infections, mainly caused by Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis are among the leading causes of global mortality and morbidity. Increased resistance of these pathogens to existing antibiotics necessitates the search for novel targets to develop potent antimicrobials. RESULT: Here, we report a proof of concept study for the reliable identification of potential drug targets in these human respiratory pathogens by combining high-density transposon mutagenesis, high-throughput sequencing, and integrative genomics. Approximately 20% of all genes in these three species were essential for growth and viability, including 128 essential and conserved genes, part of 47 metabolic pathways. By comparing these essential genes to the human genome, and a database of genes from commensal human gut microbiota, we identified and excluded potential drug targets in respiratory tract pathogens that will have off-target effects in the host, or disrupt the natural host microbiota. We propose 249 potential drug targets, 67 of which are targets for 75 FDA-approved antimicrobials and 35 other researched small molecule inhibitors. Two out of four selected novel targets were experimentally validated, proofing the concept. CONCLUSION: Here we have pioneered an attempt in systematically combining the power of high-density transposon mutagenesis, high-throughput sequencing, and integrative genomics to discover potential drug targets at genome-scale. By circumventing the time-consuming and expensive laboratory screens traditionally used to select potential drug targets, our approach provides an attractive alternative that could accelerate the much needed discovery of novel antimicrobials.

Genetic requirements for Moraxella catarrhalis growth under iron-limiting conditions.

Iron sequestration by the human host is a first line defence against respiratory pathogens like Moraxella catarrhalis, which consequently experiences a period of iron starvation during colonization. We determined the genetic requirements for M. catarrhalis BBH18 growth during iron starvation using the high-throughput genome-wide screening technology genomic array footprinting (GAF). By subjecting a large random transposon mutant library to growth under iron-limiting conditions, mutants of the MCR_0996-rhlB-yggW operon, rnd, and MCR_0457 were negatively selected. Growth experiments using directed mutants confirmed the GAF phenotypes with ΔyggW (putative haem-shuttling protein) and ΔMCR_0457 (hypothetical protein) most severely attenuated during iron starvation, phenotypes which were restored upon genetic complementation of the deleted genes. Deletion of yggW resulted in similar attenuated phenotypes in three additional strains. Transcriptional profiles of ΔyggW and ΔMCR_0457 were highly altered with 393 and 192 differentially expressed genes respectively. In all five mutants, expression of nitrate reductase genes was increased and of nitrite reductase decreased, suggesting an impaired aerobic respiration. Alteration of iron metabolism may affect nasopharyngeal colonization as adherence of all mutants to respiratory tract epithelial cells was attenuated. In conclusion, we elucidated the genetic requirements for M. catarrhalis growth during iron starvation and characterized the roles of the identified genes in bacterial growth and host interaction.

A single amino acid substitution in the MurF UDP-MurNAc-pentapeptide synthetase renders Streptococcus pneumoniae dependent on CO2 and temperature.

The respiratory tract pathogen Streptococcus pneumoniae encounters different levels of environmental CO2 during transmission, host colonization and disease. About 8% of all pneumococcal isolates are capnophiles that require CO2 -enriched growth conditions. The underlying molecular mechanism for caphnophilic behaviour, as well as its biological function is unknown. Here, we found that capnophilic S. pneumoniae isolates from clonal complex (CC) 156 (i.e. Spain(9V) -3 ancestry) and CC344 (i.e. Norway(NT) -42 ancestry) have a valine at position 179 in the MurF UDP-MurNAc-pentapeptide synthetase. At ≤ 30°C, the growth characteristics of capnophilic and non-capnophilic CC156 strains were equal, but at > 30°C growth and survival of MurF(V) (179) strains was dependent on > 0.1% CO2 -enriched conditions. Expression of MurF(V) (179) in S. pneumoniae R6 and G54 rendered these, otherwise non-capnophilic strains, capnophilic. Time-lapse microscopy revealed that a capnophilic CC156 strain undergoes rapid autolysis upon exposure to CO2 -poor conditions at 37°C, and staining with fluorescently labelled vancomycin showed a defect in de novo cell wall synthesis. In summary, in capnophilic S. pneumoniae strains from CC156 and CC344 cell wall synthesis is placed under control of environmental CO2 levels and temperature. This mechanism might represent a novel strategy of the pneumococcus to rapidly adapt and colonize its host under changing environmental conditions.

Nontypeable Haemophilus influenzae carbonic anhydrase is important for environmental and intracellular survival.

Nontypeable Haemophilus influenzae (NTHi) is one of the leading causes of noninvasive mucosal infections, such as otitis media, sinusitis, and conjunctivitis. During its life cycle, NTHi is exposed to different CO2 levels, which vary from ∼0.04% in ambient air during transmission to a new host to over 5% in the respiratory tract and tissues of the human host during colonization and disease. We used the next-generation sequencing Tn-seq technology to identify genes essential for NTHi adaptation to changes in environmental CO2 levels. It appeared that H. influenzae carbonic anhydrase (HICA), which catalyzes the reversible hydration of CO2 to bicarbonate, is a molecular factor that is conditionally essential for NTHi survival in ambient air. Growth of NTHi Δcan strains was restored under 5% CO2-enriched conditions, by supplementation of the growth medium with sodium bicarbonate, or by genetic complementation with the can gene. Finally, we showed that HICA not only is essential for environmental survival but also appeared to be important for intracellular survival in host cells. Hence, HICA is important for NTHi niche adaptation.

Streptococcus pneumoniae folate biosynthesis responds to environmental CO2 levels.

Although carbon dioxide (CO2) is known to be essential for Streptococcus pneumoniae growth, it is poorly understood how this respiratory tract pathogen adapts to the large changes in environmental CO2 levels it encounters during transmission, host colonization, and disease. To identify the molecular mechanisms that facilitate pneumococcal growth under CO2-poor conditions, we generated a random S. pneumoniae R6 mariner transposon mutant library representing mutations in 1,538 different genes and exposed it to CO2-poor ambient air. With Tn-seq, we found mutations in two genes that were involved in S. pneumoniae adaptation to changes in CO2 availability. The gene pca, encoding pneumococcal carbonic anhydrase (PCA), was absolutely essential for S. pneumoniae growth under CO2-poor conditions. PCA catalyzes the reversible hydration of endogenous CO2 to bicarbonate (HCO3(-)) and was previously demonstrated to facilitate HCO3(-)-dependent fatty acid biosynthesis. The gene folC that encodes the dihydrofolate/folylpolyglutamate synthase was required at the initial phase of bacterial growth under CO2-poor culture conditions. FolC compensated for the growth-phase-dependent decrease in S. pneumoniae intracellular long-chain (n > 3) polyglutamyl folate levels, which was most pronounced under CO2-poor growth conditions. In conclusion, S. pneumoniae adaptation to changes in CO2 availability involves the retention of endogenous CO2 and the preservation of intracellular long-chain polyglutamyl folate pools.

Combination of pantothenamides with vanin inhibitors as a novel antibiotic strategy against gram-positive bacteria.

The emergence of resistance against current antibiotics calls for the development of new compounds to treat infectious diseases. Synthetic pantothenamides are pantothenate analogs that possess broad-spectrum antibacterial activity in vitro in minimal media. Pantothenamides were shown to be substrates of the bacterial coenzyme A (CoA) biosynthetic pathway, causing cellular CoA depletion and interference with fatty acid synthesis. In spite of their potential use and selectivity for bacterial metabolic routes, these compounds have never made it to the clinic. In the present study, we show that pantothenamides are not active as antibiotics in the presence of serum, and we found that they were hydrolyzed by ubiquitous pantetheinases of the vanin family. To address this further, we synthesized a series of pantetheinase inhibitors based on a pantothenate scaffold that inhibited serum pantetheinase activity in the nanomolar range. Mass spectrometric analysis showed that addition of these pantetheinase inhibitors prevented hydrolysis of pantothenamides by serum. We found that combinations of these novel pantetheinase inhibitors and prototypic pantothenamides like N5-Pan and N7-Pan exerted antimicrobial activity in vitro, particularly against Gram-positive bacteria (Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, and Streptococcus pyogenes) even in the presence of serum. These results indicate that pantothenamides, when protected against degradation by host pantetheinases, are potentially useful antimicrobial agents.

Vaccination with SesC decreases Staphylococcus epidermidis biofilm formation.

The increased use of medical implants has resulted in a concomitant rise in device-related infections. The majority of these infections are caused by Staphylococcus epidermidis biofilms. Immunoprophylaxis and immunotherapy targeting in vivo-expressed, biofilm-associated, bacterial cell surface-exposed proteins are promising new approaches to prevent and treat biofilm-related infections, respectively. Using an in silico procedure, we identified 64 proteins that are predicted to be S. epidermidis surface exposed (Ses), of which 36 were annotated as (conserved) hypothetical. Of these 36 proteins, 5 proteins-3 LPXTG motif-containing proteins (SesL, SesB, and SesC) and 2 of the largest ABC transporters (SesK and SesM)-were selected for evaluation as vaccine candidates. This choice was based on protein size, number of antigenic determinants, or the established role in S. epidermidis biofilm formation of the protein family to which the candidate protein belongs. Anti-SesC antibodies exhibited the greatest inhibitory effect on S. epidermidis biofilm formation in vitro and on colonization and infection in a mouse jugular vein catheter infection model that includes biofilms and organ infections. Active vaccination with a recombinant truncated SesC inhibited S. epidermidis biofilm formation in a rat model of subcutaneous foreign body infection. Antibodies to SesC were shown to be opsonic by an in vitro opsonophagocytosis assay. We conclude that SesC is a promising target for antibody mediated strategies against S. epidermidis biofilm formation.

Inhibition of the ß-carbonic anhydrase from Streptococcus pneumoniae by inorganic anions and small molecules: Toward innovative drug design of antiinfectives?

The Gram-positive bacterium Streptococcus pneumoniae is a human respiratory tract pathogen that contributes significantly to global mortality and morbidity. It was recently shown that this bacterial pathogen depends on a conserved ß-carbonic anhydrase (CA, EC 4.2.1.1) for in vitro growth in environmental ambient air and during intracellular survival in host cells. Hence, it is to be expected that this pneumococcal carbonic anhydrase (PCA) contributes to transmission and pathogenesis of the bacterium, making it a potential therapeutic target. In this study, purified recombinant PCA has been further characterized kinetically and for inhibition with a series of inorganic anions and small molecules useful as leads. PCA has appreciable activity as catalyst for the hydration of CO(2) to bicarbonate, with a k(cat) of 7.4×10(5)s(-1) and k(cat)/K(m) of 6.5×10(7) M(-1)s(-1) at an optimum pH of 8.4. Inorganic anions such as chloride, bromide, iodide, cyanate, selenocyanate, trithiocarbonate, and cyanide were effective inhibitors of PCA (K(I)s of 21-98µM). Sulfamide, sulfamic acid, phenylboronic, phenylarsonic acid, and diethyldithiocarbamate showed inhibition constants in the low micromolar/submicromolar range (K(I)s of 0.61-6.68µM), whereas that of the sulfonamide acetazolamide was in the nanomolar range (K(I)s 89nM). In conclusion, our results show that PCA can effectively be inhibited by a range of molecules that could be interesting leads for obtaining more potent PCA inhibitors. PCA might be a novel target for designing antimicrobial drugs with a new mechanism of action.

Carbonic anhydrase is essential for Streptococcus pneumoniae growth in environmental ambient air.

The respiratory tract pathogen Streptococcus pneumoniae needs to adapt to the different levels of carbon dioxide (CO(2)) it encounters during transmission, colonization, and infection. Since CO(2) is important for various cellular processes, factors that allow optimal CO(2) sequestering are likely to be important for pneumococcal growth and survival. In this study, we showed that the putative pneumococcal carbonic anhydrase (PCA) is essential for in vitro growth of S. pneumoniae under the CO(2)-poor conditions found in environmental ambient air. Enzymatic analysis showed that PCA catalyzes the reversible hydration of CO(2) to bicarbonate (HCO(3)(-)), an essential step to prevent the cellular release of CO(2). The addition of unsaturated fatty acids (UFAs) reversed the CO(2)-dependent in vitro growth inhibition of S. pneumoniae strains lacking the pca gene (Deltapca), indicating that PCA-mediated CO(2) fixation is at least associated with HCO(3)(-)-dependent de novo biosynthesis of UFAs. Besides being necessary for growth in environmental ambient conditions, PCA-mediated CO(2) fixation pathways appear to be required for intracellular survival in host cells. This effect was especially pronounced during invasion of human brain microvascular endothelial cells (HBMEC) and uptake by murine J774 macrophage cells but not during interaction of S. pneumoniae with Detroit 562 pharyngeal epithelial cells. Finally, the highly conserved pca gene was found to be invariably present in both CO(2)-independent and naturally circulating CO(2)-dependent strains, suggesting a conserved essential role for PCA and PCA-mediated CO(2) fixation pathways for pneumococcal growth and survival.

spr1630 is responsible for the lethality of clpX mutations in Streptococcus pneumoniae.

The Clp protease ATPase subunit and chaperone ClpX is dispensable in some bacteria, but it is thought to be essential in others, including streptococci and lactococci. We confirm that clpX is essential in the Rx strain of Streptococcus pneumoniae but show that the requirement for clpX can be relieved by point mutations, frame shifts, or deletion of the gene spr1630, which is found in many isolates of S. pneumoniae. Homologs occur frequently in Staphylococcus aureus as well as in a few strains of Listeria monocytogenes, Lactobacillus johnsonii, and Lactobacillus rhamnosus. In each case, the spr1630 homolog is accompanied by a putative transcriptional regulator with an HTH DNA binding motif. In S. pneumoniae, the spr1630-spr1629 gene pair, accompanied by a RUP element, occurs as an island inserted between the trpA and cclA genes in 15 of 22 sequenced genomes.

Genome-wide identification of genes essential for the survival of Streptococcus pneumoniae in human saliva.

Since Streptococcus pneumoniae transmits through droplet spread, this respiratory tract pathogen may be able to survive in saliva. Here, we show that saliva supports survival of clinically relevant S. pneumoniae strains for more than 24 h in a capsule-independent manner. Moreover, saliva induced growth of S. pneumoniae in growth-permissive conditions, suggesting that S. pneumoniae is well adapted for uptake of nutrients from this bodily fluid. By using Tn-seq, a method for genome-wide negative selection screening, we identified 147 genes potentially required for growth and survival of S. pneumoniae in saliva, among which genes predicted to be involved in cell envelope biosynthesis, cell transport, amino acid metabolism, and stress response predominated. The Tn-seq findings were validated by testing a panel of directed gene deletion mutants for their ability to survive in saliva under two testing conditions: at room temperature without CO2, representing transmission, and at 37 °C with CO2, representing in-host carriage. These validation experiments confirmed that the plsX gene and the amiACDEF and aroDEBC operons, involved in respectively fatty acid metabolism, oligopeptide transport, and biosynthesis of aromatic amino acids play an important role in the growth and survival of S. pneumoniae in saliva at 37 °C. In conclusion, this study shows that S. pneumoniae is well-adapted for growth and survival in human saliva and provides a genome-wide list of genes potentially involved in adaptation. This notion supports earlier evidence that S. pneumoniae can use human saliva as a vector for transmission.

ESSENTIALS: software for rapid analysis of high throughput transposon insertion sequencing data.

High-throughput analysis of genome-wide random transposon mutant libraries is a powerful tool for (conditional) essential gene discovery. Recently, several next-generation sequencing approaches, e.g. Tn-seq/INseq, HITS and TraDIS, have been developed that accurately map the site of transposon insertions by mutant-specific amplification and sequence readout of DNA flanking the transposon insertions site, assigning a measure of essentiality based on the number of reads per insertion site flanking sequence or per gene. However, analysis of these large and complex datasets is hampered by the lack of an easy to use and automated tool for transposon insertion sequencing data. To fill this gap, we developed ESSENTIALS, an open source, web-based software tool for researchers in the genomics field utilizing transposon insertion sequencing analysis. It accurately predicts (conditionally) essential genes and offers the flexibility of using different sample normalization methods, genomic location bias correction, data preprocessing steps, appropriate statistical tests and various visualizations to examine the results, while requiring only a minimum of input and hands-on work from the researcher. We successfully applied ESSENTIALS to in-house and published Tn-seq, TraDIS and HITS datasets and we show that the various pre- and post-processing steps on the sequence reads and count data with ESSENTIALS considerably improve the sensitivity and specificity of predicted gene essentiality.

Nonencapsulated Streptococcus pneumoniae resists extracellular human neutrophil elastase- and cathepsin G-mediated killing.

Although the Streptococcus pneumoniae polysaccharide capsule is an important virulence factor, ~ 15% of carriage isolates are nonencapsulated. Nonencapsulated S. pneumoniae are a cause of mucosal infections. Recent studies have shown that neutrophils kill S. pneumoniae predominately through neutrophil proteases, such as elastase and cathepsin G. Another recent finding is that nonencapsulated pneumococci have greater resistance to resist cationic antimicrobial peptides that are important in mucosal immunity. We here show that nonencapsulated pneumococci have greater resistance to extracellular human neutrophil elastase- and cathepsin G-mediated killing than isogenic encapsulated pneumococci. Resistance to extracellular neutrophil protease-mediated killing is likely to be of greater relative importance on mucosal surfaces compared to other body sites.

Modified lipooligosaccharide structure protects nontypeable Haemophilus influenzae from IgM-mediated complement killing in experimental otitis media.

UNLABELLED: Nontypeable Haemophilus influenzae (NTHi) is a Gram-negative, human-restricted pathogen. Although this bacterium typically colonizes the nasopharynx in the absence of clinical symptoms, it is also one of the major pathogens causing otitis media (OM) in children. Complement represents an important aspect of the host defense against NTHi. In general, NTHi is efficiently killed by complement-mediated killing; however, various resistance mechanisms have also evolved. We measured the complement resistance of NTHi isolates isolated from the nasopharynx and the middle ear fluids of OM patients. Furthermore, we determined the molecular mechanism of NTHi complement resistance. Complement resistance was strongly increased in isolates from the middle ear, which correlated with decreased binding of IgM. We identified a crucial role for the R2866_0112 gene in complement resistance. Deletion of this gene altered the lipooligosaccharide (LOS) composition of the bacterium, which increased IgM binding and complement-mediated lysis. In a novel mouse model of coinfection with influenza virus, we demonstrate decreased virulence for the R2866_0112 deletion mutant. These findings identify a mechanism by which NTHi modifies its LOS structure to prevent recognition by IgM and activation of complement. Importantly, this mechanism plays a crucial role in the ability of NTHi to cause OM. IMPORTANCE: Nontypeable Haemophilus influenzae (NTHi) colonizes the nasopharynx of especially young children without any obvious symptoms. However, NTHi is also a major pathogen in otitis media (OM), one of the most common childhood infections. Although this pathogen is often associated with OM, the mechanism by which this bacterium is able to cause OM is largely unknown. Our study addresses a key biological question that is highly relevant for child health: what is the molecular mechanism that enables NTHi to cause OM? We show that isolates collected from the middle ear fluid exhibit increased complement resistance and that the lipooligosaccharide (LOS) structure determines IgM binding and complement activation. Modification of the LOS structure decreased NTHi virulence in a novel NTHi-influenza A virus coinfection OM mouse model. Our findings may also have important implications for other Gram-negative pathogens harboring LOS, such as Neisseria meningitidis, Moraxella catarrhalis, and Bordetella pertussis.

Development of genomic array footprinting for identification of conditionally essential genes in Streptococcus pneumoniae.

Streptococcus pneumoniae is a major cause of serious infections such as pneumonia and meningitis in both children and adults worldwide. Here, we describe the development of a high-throughput, genome-wide technique, genomic array footprinting (GAF), for the identification of genes essential for this bacterium at various stages during infection. GAF enables negative screens by means of a combination of transposon mutagenesis and microarray technology for the detection of transposon insertion sites. We tested several methods for the identification of transposon insertion sites and found that amplification of DNA adjacent to the insertion site by PCR resulted in nonreproducible results, even when combined with an adapter. However, restriction of genomic DNA followed directly by in vitro transcription circumvented these problems. Analysis of parallel reactions generated with this method on a large mariner transposon library showed that it was highly reproducible and correctly identified essential genes. Comparison of a mariner library to one generated with the in vivo transposition plasmid pGh:ISS1 showed that both have an equal degree of saturation but that 9% of the genome is preferentially mutated by either one. The usefulness of GAF was demonstrated in a screen for genes essential for surviving zinc stress. This identified a gene encoding a putative cation efflux transporter, and its deletion resulted in an inability to grow under high-zinc conditions. In conclusion, we developed a fast, versatile, specific, and high-throughput method for the identification of conditionally essential genes in S. pneumoniae.

Search for genes essential for pneumococcal transformation: the RADA DNA repair protein plays a role in genomic recombination of donor DNA.

We applied a novel negative selection strategy called genomic array footprinting (GAF) to identify genes required for genetic transformation of the gram-positive bacterium Streptococcus pneumoniae. Genome-wide mariner transposon mutant libraries in S. pneumoniae strain R6 were challenged by transformation with an antibiotic resistance cassette and growth in the presence of the corresponding antibiotic. The GAF screen identified the enrichment of mutants in two genes, i.e., hexA and hexB, and the counterselection of mutants in 21 different genes during the challenge. Eight of the counterselected genes were known to be essential for pneumococcal transformation. Four other genes, i.e., radA, comGF, parB, and spr2011, have previously been linked to the competence regulon, and one, spr2014, was located adjacent to the essential competence gene comFA. Directed mutants of seven of the eight remaining genes, i.e., spr0459-spr0460, spr0777, spr0838, spr1259-spr1260, and spr1357, resulted in reduced, albeit modest, transformation rates. No connection to pneumococcal transformation could be made for the eighth gene, which encodes the response regulator RR03. We further demonstrated that the gene encoding the putative DNA repair protein RadA is required for efficient transformation with chromosomal markers, whereas transformation with replicating plasmid DNA was not significantly affected. The radA mutant also displayed an increased sensitivity to treatment with the DNA-damaging agent methyl methanesulfonate. Hence, RadA is considered to have a role in recombination of donor DNA and in DNA damage repair in S. pneumoniae.

Role of the pilot protein YscW in the biogenesis of the YscC secretin in Yersinia enterocolitica.

The YscC secretin is a major component of the type III protein secretion system of Yersinia enterocolitica and forms an oligomeric structure in the outer membrane. In a mutant lacking the outer membrane lipoprotein YscW, secretion is strongly reduced, and it has been proposed that YscW plays a role in the biogenesis of the secretin. To study the interaction between the secretin and this putative pilot protein, YscC and YscW were produced in trans in a Y. enterocolitica strain lacking all other components of the secretion machinery. YscW expression increased the yield of oligomeric YscC and was required for its outer membrane localization, confirming the function of YscW as a pilot protein. Whereas the pilot-binding site of other members of the secretin family has been identified in the C terminus, a truncated YscC derivative lacking the C-terminal 96 amino acid residues was functional and stabilized by YscW. Pulse-chase experiments revealed that approximately 30 min were required before YscC oligomerization was completed. In the absence of YscW, oligomerization was delayed and the yield of YscC oligomers was strongly reduced. An unlipidated form of the YscW protein was not functional, although it still interacted with the secretin and caused mislocalization of YscC even in the presence of wild-type YscW. Hence, YscW interacts with the unassembled YscC protein and facilitates efficient oligomerization, likely at the outer membrane.

Structure and electrophysiological properties of the YscC secretin from the type III secretion system of Yersinia enterocolitica.

YscC is the integral outer membrane component of the type III protein secretion machinery of Yersinia enterocolitica and belongs to the family of secretins. This group of proteins forms stable ring-like oligomers in the outer membrane, which are thought to function as transport channels for macromolecules. The YscC oligomer was purified after solubilization from the membrane with a nonionic detergent. Sodium dodecyl sulfate did not dissociate the oligomer, but it caused a change in electrophoretic mobility and an increase in protease susceptibility, indicating partial denaturation of the subunits within the oligomer. The mass of the homo-oligomer, as determined by scanning transmission electron microscopy, was approximately 1 MDa. Analysis of the angular power spectrum from averaged top views of negatively stained YscC oligomers revealed a 13-fold angular order, suggesting that the oligomer consists of 13 subunits. Reconstituted in planar lipid bilayers, the YscC oligomer displayed a constant voltage-independent conductance of approximately 3 nS, thus forming a stable pore. However, in vivo, the expression of YscC did not lead to an increased permeability of the outer membrane. Electron microscopy revealed that the YscC oligomer is composed of three domains, two stacked rings attached to a conical domain. This structure is consistent with the notion that the secretin forms the upper part of the basal body of the needle structure of the type III secreton.