Various gram-negative animal and plant pathogens use a novel, sec-independent protein secretion system as a basic virulence mechanism. It is becoming increasingly clear that these so-called type III secretion systems inject (translocate) proteins into the cytosol of eukaryotic cells, where the translocated proteins facilitate bacterial pathogenesis by specifically interfering with host cell signal transduction and other cellular processes. Accordingly, some type III secretion systems are activated by bacterial contact with host cell surfaces. Individual type III secretion systems direct the secretion and translocation of a variety of unrelated proteins, which account for species-specific pathogenesis phenotypes. In contrast to the secreted virulence factors, most of the 15 to 20 membrane-associated proteins which constitute the type III secretion apparatus are conserved among different pathogens. Most of the inner membrane components of the type III secretion apparatus show additional homologies to flagellar biosynthetic proteins, while a conserved outer membrane factor is similar to secretins from type II and other secretion pathways. Structurally conserved chaperones which specifically bind to individual secreted proteins play an important role in type III protein secretion, apparently by preventing premature interactions of the secreted factors with other proteins. The genes encoding type III secretion systems are clustered, and various pieces of evidence suggest that these systems have been acquired by horizontal genetic transfer during evolution. Expression of type III secretion systems is coordinately regulated in response to host environmental stimuli by networks of transcription factors. This review comprises a comparison of the structure, function, regulation, and impact on host cells of the type III secretion systems in the animal pathogens Yersinia spp., Pseudomonas aeruginosa, Shigella flexneri, Salmonella typhimurium, enteropathogenic Escherichia coli, and Chlamydia spp. and the plant pathogens Pseudomonas syringae, Erwinia spp., Ralstonia solanacearum, Xanthomonas campestris, and Rhizobium spp.

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Type III Protein Secretion Systems in Bacterial Pathogens of Animals and Plants

Twitching motility is a flagella-independent form of bacterial translocation over moist surfaces. It occurs by the extension, tethering, and then retraction of polar type IV pili, which operate in a manner similar to a grappling hook. Twitching motility is equivalent to social gliding motility in Myxococcus xanthus and is important in host colonization by a wide range of plant and animal pathogens, as well as in the formation of biofilms and fruiting bodies. The biogenesis and function of type IV pili is controlled by a large number of genes, almost 40 of which have been identified in Pseudomonas aeruginosa. A number of genes required for pili assembly are homologous to genes involved in type II protein secretion and competence for DNA uptake, suggesting that these systems share a common architecture. Twitching motility is also controlled by a range of signal transduction systems, including two-component sensor-regulators and a complex chemosensory system.

Type IV pili and twitching motility.

Antimicrobial factors form one arm of the innate immune system, which protects mucosal surfaces from bacterial infection. These factors can rapidly kill bacteria deposited on mucosal surfaces and prevent acute invasive infections. In many chronic infections, however, bacteria live in biofilms, which are distinct, matrix-encased communities specialized for surface persistence. The transition from a free-living, independent existence to a biofilm lifestyle can be devastating, because biofilms notoriously resist killing by host defence mechanisms and antibiotics. We hypothesized that the innate immune system possesses specific activity to protect against biofilm infections. Here we show that lactoferrin, a ubiquitous and abundant constituent of human external secretions, blocks biofilm development by the opportunistic pathogen Pseudomonas aeruginosa. This occurs at lactoferrin concentrations below those that kill or prevent growth. By chelating iron, lactoferrin stimulates twitching, a specialized form of surface motility, causing the bacteria to wander across the surface instead of forming cell clusters and biofilms. These findings reveal a specific anti-biofilm defence mechanism acting at a critical juncture in biofilm development, the time bacteria stop roaming as individuals and aggregate into durable communities.

A component of innate immunity prevents bacterial biofilm development

Structures in Other Domains The methodology of structural analysis discussed in this article has been applied beyond the narrow realm of natural language syntax that we have discussed in this article. Within the study of language, similar methods of analysis have been pervasively applied to the study of sounds (phonology), words (morphology), and meanings (semantics), yielding a range of of abstract structural representations whose properties bear considerable explanatory burden. There are a wealth of cases in each of these domains analogous to those discussed here, though space prevents us from going in these (see Akmajian, Demers, Farmer and Harnish 1995 for a traditional overview, and Jackendo 1994 for one more focused on connections with cognitive science). Additionally, these representations have shed substantial light on the processes of language acquisition and language change. It has been found that variation in the types of sentences that are used, whether during the course of children's acquisition of their native languages or in the centuries-long periods of linguistic change, are best characterized not as super cial and haphazard alterations, but rather in terms of parametric modi cations to the fundamental underlying grammatical rules and constraints. Moving outside the domain of language, one application of these same methods has been in the study of music cognition. Just as the representations of linguistic theory arise out of an attempt to model speakers' intuitions about well-formedness and possible meanings of the sentences of their

Structural analysis

Natural competence is widespread among bacterial species. The mechanism of DNA uptake in both gram-positive and gram-negative bacteria is reviewed. The transformation pathways are discussed, with attention to the fate of donor DNA as it is processed by the competent cell. The proteins involved in mediating various steps in these pathways are described, and models for the transformation mechanisms are presented. Uptake of DNA across the inner membrane is probably similar in gram-positive and gram-negative bacteria, and at least some of the required proteins are orthologs. The initial transformation steps differ, as expected, from the presence of an outer membrane only in the gram-negative organisms. The similarity of certain essential competence proteins to those required for the assembly of type-4 pili and for type-2 protein secretion is discussed. Finally several hypotheses for the biological role of transformation are presented and evaluated.

DNA Uptake in Bacteria

Virulence of the opportunistic pathogen Pseudomonas aeruginosa involves the coordinate expression of a wide range of virulence factors including type IV pili which are required for colonization of host tissues and are associated with a form of surface translocation termed twitching motility. Twitching motility in P. aeruginosa is controlled by a complex signal transduction pathway which shares many modules in common with chemosensory systems controlling flagella rotation in bacteria and which is composed, in part, of the previously described proteins PilG, PilH, PilI, PilJ and PilK. Here we describe another three components of this pathway: ChpA, ChpB and ChpC, as well as two downstream genes, ChpD and ChpE, which may also be involved. The central component of the pathway, ChpA, possesses nine potential sites of phosphorylation: six histidine-containing phosphotransfer (HPt) domains, two novel serine- and threonine-containing phosphotransfer (SPt, TPt) domains and a CheY-like receiver domain at its C-terminus, and as such represents one of the most complex signalling proteins yet described in nature. We show that the Chp chemosensory system controls twitching motility and type IV pili biogenesis through control of pili assembly and/or retraction as well as expression of the pilin subunit gene pilA. The Chp system is also required for full virulence in a mouse model of acute pneumonia.

Characterization of a complex chemosensory signal transduction system which controls twitching motility in Pseudomonas aeruginosa

Type 4 fimbriae are produced by a variety of pathogens, in which they appear to function in adhesion to epithelial cells, and in a form of surface translocation called twitching motility. Using transposon mutagenesis of Pseudomonas aeruginosa, we have identified a new locus required for fimbrial assembly. This locus contains the gene pilQ which encodes a 77 kDa protein with an N-terminal hydrophobic signal sequence characteristic of secretory proteins. pilQ mutants lack the spreading colony morphology characteristic of twitching motility, are devoid of fimbriae, and are resistant to the fimbrial-specific bacteriophage PO4. The pilQ gene was mapped to Spel fragment 2, which is located at 0-5 minutes on the P. aeruginosa PAO1 chromosome, and thus it is not closely linked to the previously characterized pilA-D, pilS,R or pilT genes. The pilQ region also contains ponA, aroK and aroB-like genes in an organization very similar to that of corresponding genes in Escherichia coli and Haemophilus influenzae. The predicted amino acid sequence of PilQ shows homology to the PulD protein of Klebsiella oxytoca and related outer membrane proteins which have been found in association with diverse functions in other species including protein secretion, DNA uptake and assembly of filamentous phage. PilQ had the highest overall homology to an outer membrane antigen from Neisseria gonorrhoeae, encoded by omc, that may fulfil the same role in type 4 fimbrial assembly in this species.

Characterization of pilQ, a new gene required for the biogenesis of type 4 fimbriae in Pseudomonas aeruginosa.

The opportunistic pathogen Pseudomonas aeruginosa produces type 4 fimbriae which promote adhesion to epithelial cells and are associated with a form of surface translocation called twitching motility. Transposon mutagenesis was used to identify loci required for fimbrial assembly or function by screening for mutants that lack the spreading colony morphology characteristic of twitching motility. Six mutants were isolated that contain transposon insertions upstream of the previously characterized gene pilQ. This region contains four genes: pilM-P, which encode proteins with predicted sizes of 37.9, 22.2, 22.8 and 19.0 kDa, respectively. pilM-P appear to form an operon and to be expressed from a promoter in the intergenic region between pilM and the divergently transcribed upstream gene ponA. PilM-P were found to be required for fimbrial biogenesis by complementation studies using twitching motility and sensitivity to fimbrial-specific phage as indicators of the presence of functional fimbriae. This was confirmed by electron microscopy. PilO and PilP did not have homologues in the sequence databases, but the predicted PilN amino acid sequence displayed similarity to XpsL from Xanthamonas campestris, a protein required for protein secretion. PilP contained a hydrophobic leader sequence characteristic of lipoproteins, while PilN and PilO have long internal hydrophobic domains which may serve to localize them to the cytoplasmic membrane. PilM has shared sequence motifs with the cell division protein FtsA from Bacillus subtilis and Escherichia coli, as well as the rod-shape-determining protein MreB from E. coli. These motifs are also conserved in eukaryotic actin, in which they are involved in forming an ATPase domain. Deletion mutants of pilM and pilQ displayed a dominant negative phenotype when transformed into wild-type cells, suggesting that these genes encode proteins involved in multimeric structures.

Paul R. Martin

Papers

Characterization of a complex chemosensory signal transduction system which controls twitching motility in Pseudomonas aeruginosa

Characterization of pilQ, a new gene required for the biogenesis of type 4 fimbriae in Pseudomonas aeruginosa.

Characterization of a five-gene cluster required for the biogenesis of type 4 fimbriae in Pseudomonas aeruginosa.