SawR a new regulator controlling pyomelanin synthesis in Pseudomonas aeruginosa.

Surface Acoustic Waves (SAW) were previously shown to inhibit biofilm formation, increase bacterial susceptibility to antibiotic treatment and alter the transcription pattern of Pseudomonas aeruginosa. Here we characterize one gene, sawR (PA3133), that is highly overexpressed when P. aeruginosa is exposed to SAW. SawR is a putative transcription factor belonging to the TetR regulator family. When overexpressed sawR causes numerous phenotypes, including the accumulation of a brown pigment which we identified as pyomelanin. In this study we describe how sawR regulates pyomelanin synthesis. We show that sawR down-regulates the expression levels of hmgA and this causes the accumulation of homogentisic acid which in turn undergoes oxidation and polymerization to pyomelanin. Using bioinformatics, we were able to identify a specific amino acid, arginine 23, which is found within the sawR DNA binding domain and is crucial for its regulatory activity. Our results indicate that sawR does not affect any other genes in the phenylalanine/tyrosine metabolic pathway and its repressive ability on hmgA is not mediated by the hmgA repressor PA2010 (i.e. hmgR). Taken together, our results shed light on the regulatory cascade controlling pyomelanin synthesis and uncover yet another unknown regulator involved in its regulation.

Novel type III effectors in Pseudomonas aeruginosa.

UNLABELLED: Pseudomonas aeruginosa is a Gram-negative, opportunistic pathogen that causes chronic and acute infections in immunocompromised patients. Most P. aeruginosa strains encode an active type III secretion system (T3SS), utilized by the bacteria to deliver effector proteins from the bacterial cell directly into the cytoplasm of the host cell. Four T3SS effectors have been discovered and extensively studied in P. aeruginosa: ExoT, ExoS, ExoU, and ExoY. This is especially intriguing in light of P. aeruginosa's ability to infect a wide range of hosts. We therefore hypothesized that additional T3SS effectors that have not yet been discovered are encoded in the genome of P. aeruginosa. Here, we applied a machine learning classification algorithm to identify novel P. aeruginosa effectors. In this approach, various types of data are integrated to differentiate effectors from the rest of the open reading frames of the bacterial genome. Due to the lack of a sufficient learning set of positive effectors, our machine learning algorithm integrated genomic information from another Pseudomonas species and utilized dozens of features accounting for various aspects of the effector coding genes and their products. Twelve top-ranking predictions were experimentally tested for T3SS-specific translocation, leading to the discovery of two novel T3SS effectors. We demonstrate that these effectors are not part of the injection structural complex and report initial efforts toward their characterization. IMPORTANCE: Pseudomonas aeruginosa uses a type III secretion system (T3SS) to secrete toxic proteins, termed effectors, directly into the cytoplasm of the host cell. The activation of this secretion system is correlated with disease severity and patient death. Compared with many other T3SS-utilizing pathogenic bacteria, P. aeruginosa has a fairly limited arsenal of effectors that have been identified. This is in sharp contrast with the wide range of hosts that this bacterium can infect. The discovery of two novel effectors described here is an important step toward better understanding of the virulence and host evasion mechanisms adopted by this versatile pathogen and may provide novel approaches to treat P. aeruginosa infections.

The Pseudomonas aeruginosa phosphate transport protein PstS plays a phosphate-independent role in biofilm formation.

Pseudomonas aeruginosa (PA) is a primary cause of nosocomial infections. A key element in PA pathogenicity is its ability to form biofilms that withstand eradication by antibiotics and the immune system. Biofilm formation is controlled by phosphate signaling and here we provide evidence that PstS, a subunit of the PA Pst phosphate transporter, has a surprising role in this process. Using X-ray crystallography, we characterized the unique underpinnings of PstS phosphate binding and identified an unusual 15-residue N' loop extension. Structure-based experiments showed that PstS-mediated phosphate uptake and biofilm formation are in fact two distinct functions. Specifically, a point mutation that abrogated phosphate binding did not eliminate biofilm formation; conversely, truncation of the N' loop diminished the ability of PA to form biofilms but had no effect on phosphate binding and uptake. This places PstS at a junction that separately controls phosphate sensing and uptake and the ultrastructure organization of bacteria.

Dynamics of expression and maturation of the type III secretion system of enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) is a major cause of food poisoning, leading to significant morbidity and mortality. EPEC virulence is dependent on a type III secretion system (T3SS), a molecular syringe employed by EPEC to inject effector proteins into host cells. The injected effector proteins subvert host cellular functions to the benefit of the infecting bacteria. The T3SS and related genes reside in several operons clustered in the locus of enterocyte effacement (LEE). We carried out simultaneous analysis of the expression dynamics of all the LEE promoters and the rate of maturation of the T3SS. The results showed that expression of the LEE1 operon is activated immediately upon shifting the culture to inducing conditions, while expression of other LEE promoters is activated only ∼70 min postinduction. Parallel analysis showed that the T3SS becomes functional around 100 min postinduction. The T3SS core proteins EscS, EscT, EscU, and EscR are predicted to be involved in the first step of T3SS assembly and are therefore included among the LEE1 genes. However, interfering with the temporal regulation of EscS, EscT, EscU, and EscR expression has only a marginal effect on the rate of the T3SS assembly. This study provides a comprehensive description of the transcription dynamics of all the LEE genes and correlates it to that of T3SS biogenesis.

The effect of pstS and phoB on quorum sensing and swarming motility in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen that can cause a wide range of infections and inflammations in a variety of hosts, such as chronic biofilm associated lung infections in Cystic Fibrosis patients. Phosphate, an essential nutrient, has been recognized as an important signal that affects virulence in P. aeruginosa. In the current study we examined the connection between phosphate regulation and surface motility in P. aeruginosa. We focused on two important genes, pstS, which is involved in phosphate uptake, and phoB, a central regulator that responds to phosphate starvation. We found that a mutant lacking pstS is constantly starved for phosphate and has a hyper swarming phenotype. Phosphate starvation also induced swarming in the wild type. The phoB mutant, on the other hand, did not express phosphate starvation even when phosphate was limited and showed no swarming. A double mutant lacking both genes (pstS and phoB) showed a similar phenotype to the phoB mutant (i.e. no swarming). This highlights the role of phoB in controlling swarming motility under phosphate-depleted conditions. Finally, we were able to demonstrate that PhoB controls swarming by up-regulating the Rhl quorum sensing system in P. aeruginosa, which resulted in hyper production of rhamonlipids: biosurfactants that are known to induce swarming motility.

Metalloprotease type III effectors that specifically cleave JNK and NF-κB.

Two major arms of the inflammatory response are the NF-κB and c-Jun N-terminal kinase (JNK) pathways. Here, we show that enteropathogenic Escherichia coli (EPEC) employs the type III secretion system to target these two signalling arms by injecting host cells with two effector proteins, NleC and NleD. We provide evidence that NleC and NleD are Zn-dependent endopeptidases that specifically clip and inactivate RelA (p65) and JNK, respectively, thus blocking NF-κB and AP-1 activation. We show that NleC and NleD co-operate and complement other EPEC effectors in accomplishing maximal inhibition of IL-8 secretion. This is a remarkable example of a pathogen using multiple effectors to manipulate systematically the host inflammatory response signalling network.

Mutational analysis of the locus of enterocyte effacement-encoded regulator (Ler) of enteropathogenic Escherichia coli.

The locus of enterocyte effacement (LEE) pathogenicity island of enterohemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC, respectively) comprises a cluster of operons encoding a type III secretion system and related proteins, all of which are essential for bacterial colonization of the host intestines. The LEE1 operon encodes Ler, which positively regulates many EPEC and EHEC virulence genes located in the LEE region and elsewhere in the chromosome. In addition, Ler is a specific autorepressor of LEE1 transcription. To better understand the function of Ler, we screened for Ler mutants defective in autorepression. We isolated 18 different point mutations in Ler, rendering it defective in autorepression and in DNA binding. Among these mutants were those defective in positive regulation as well as in autorepression, dominant-negative mutants, and a mutant deficient in oligomerization. Importantly, a group of Ler autorepression mutants complemented an EPEC ler deletion mutant for transcription activation in a dosage-dependent manner, suggesting that Ler and possibly other autorepressors have an intrinsic compensatory mechanism that enables them to sustain mutations. In addition, the phenotypes of the different mutants identified by the screen define a novel domain in Ler that is required for oligomerization.

Transient shielding of intimin and the type III secretion system of enterohemorrhagic and enteropathogenic Escherichia coli by a group 4 capsule.

Enterohemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC, respectively) strains represent a major global health problem. Their virulence is mediated by the concerted activity of an array of virulence factors including toxins, a type III protein secretion system (TTSS), pili, and others. We previously showed that EPEC O127 forms a group 4 capsule (G4C), and in this report we show that EHEC O157 also produces a G4C, whose assembly is dependent on the etp, etk, and wzy genes. We further show that at early time points postinfection, these G4Cs appear to mask surface structures including intimin and the TTSS. This masking inhibited the attachment of EPEC and EHEC to tissue-cultured epithelial cells, diminished their capacity to induce the formation of actin pedestals, and attenuated TTSS-mediated protein translocation into host cells. Importantly, we found that Ler, a positive regulator of intimin and TTSS genes, represses the expression of the capsule-related genes, including etp and etk. Thus, the expression of TTSS and G4C is conversely regulated and capsule production is diminished upon TTSS expression. Indeed, at later time points postinfection, the diminishing capsule no longer interferes with the activities of intimin and the TTSS. Notably, by using the rabbit infant model, we found that the EHEC G4C is required for efficient colonization of the rabbit large intestine. Taken together, our results suggest that temporal expression of the capsule, which is coordinated with that of the TTSS, is required for optimal EHEC colonization of the host intestine.

Structure of GrlR and the implication of its EDED motif in mediating the regulation of type III secretion system in EHEC.

Enterohemorrhagic Escherichia coli (EHEC) is a common cause of severe hemorrhagic colitis. EHEC's virulence is dependent upon a type III secretion system (TTSS) encoded by 41 genes. These genes are organized in several operons clustered in the locus of enterocyte effacement. Most of the locus of enterocyte effacement genes, including grlA and grlR, are positively regulated by Ler, and Ler expression is positively and negatively modulated by GrlA and GrlR, respectively. However, the molecular basis for the GrlA and GrlR activity is still elusive. We have determined the crystal structure of GrlR at 1.9 A resolution. It consists of a typical beta-barrel fold with eight beta-strands containing an internal hydrophobic cavity and a plug-like loop on one side of the barrel. Strong hydrophobic interactions between the two beta-barrels maintain the dimeric architecture of GrlR. Furthermore, a unique surface-exposed EDED (Glu-Asp-Glu-Asp) motif is identified to be critical for GrlA-GrlR interaction and for the repressive activity of GrlR. This study contributes a novel molecular insight into the mechanism of GrlR function.

Additive effect on intracellular growth by Legionella pneumophila Icm/Dot proteins containing a lipobox motif.

Legionella pneumophila, the causative agent of Legionnaires' disease, utilizes a type IVB secretion system to subvert its host cells and grow intracellularly. This type IV secretion system is composed of 25 icm (or dot) genes that probably constitute parts of a secretion complex as well as more than 30 proteins that are translocated via this system into the host cells. Three of the Icm/Dot proteins (DotD, DotC, and IcmN) contain a lipobox motif at their N terminals and are predicted to be lipoproteins. Two of these lipoproteins (DotD and DotC) were found to be essential for intracellular growth in both HL-60-derived human macrophages and in the protozoan host Acanthamoeba castellanii, while the third lipoprotein (IcmN) was found to be partially required for intracellular growth only in A. castellanii. Mutation analysis of the lipobox cysteine residue, which was shown previously to be indispensable for the lipobox function, indicated that both DotC and DotD are partially functional without this conserved residue. Cysteine mutations in both DotC and DotD or in DotC together with an icmN deletion or in DotD together with an icmN deletion were found to be additive, indicating that each of these lipoproteins performs its function independently from the others. Analysis of the transcriptional regulation of both the dotDC operon and the icmN gene revealed that both had higher levels of expression at stationary phase which were partially dependent on the LetA regulator. Our results indicate that the lipoproteins of the L. pneumophila icm (or dot) system are essential components of the secretion system and that they perform their functions independently.

Functional similarities between the icm/dot pathogenesis systems of Coxiella burnetii and Legionella pneumophila.

Coxiella burnetii, the etiological agent of Q fever, is an obligate intracellular pathogen, whereas Legionella pneumophila, the causative agent of Legionnaires' disease, is a facultative intracellular pathogen. During infection of humans both of these pathogens multiply in alveolar macrophages inside a closed phagosome. L. pneumophila intracellular multiplication was shown to be dependent on the icm/dot system, which probably encodes a type IV-related translocation apparatus. Recently, genes homologous to all of the L. pneumophila icm/dot genes (besides icmR) were found in C. burnetii. To explore the similarities and differences between the icm/dot pathogenesis systems of these two pathogens, interspecies complementation analysis was performed. Nine C. burnetii icm homologous genes (icmT, icmS, icmQ, icmP, icmO, icmJ, icmB, icmW, and icmX) were cloned under regulation of the corresponding L. pneumophila icm genes and examined for the ability to complement L. pneumophila mutants with mutations in these genes. The C. burnetii icmS and icmW homologous genes were found to complement the corresponding L. pneumophila icm mutants to wild-type levels of intracellular growth in both HL-60-derived human macrophages and Acanthamoeba castellanii. In addition, the C. burnetii icmT homologous gene was found to completely complement an L. pneumophila insertion mutant for intracellular growth in HL-60-derived human macrophages, but it only partially complemented the same mutant for intracellular growth in A. castellanii. Moreover, as previously shown for L. pneumophila, the proteins encoded by the C. burnetii icmS and icmW homologous genes were found to interact with one another, and interspecies protein interaction was observed as well. Our results strongly indicate that the Icm/Dot pathogenesis systems of C. burnetii and L. pneumophila have common features.