Sequence Diversity of the oprI Gene,Coding for Major Outer Membrane Lipoprotein I,among rRNA Group I Pseudomonads

The sequence of oprI, the gene coding for the major outer membrane lipoprotein I, was determined by PCR sequencing for representatives of 17 species of rRNA group I pseudomonads, with a special emphasis on Pseudomonas aeruginosa and Pseudomonas fluorescens. Within the P. aeruginosa species, oprI sequences for 25 independent isolates were found to be identical, except for one silent substitution at position 96. The oprI sequences diverged more for the other rRNA group I pseudomonads (85 to 91% similarity with P. aeruginosa oprI). An accumulation of silent and also (but to a much lesser extent) nonsilent substitutions in the different sequences was found. A clustering according to the respective presence and/or positions of the HaeIII, PvuII, and SphI sites could also be obtained. A sequence cluster analysis showed a rather widespread distribution of P. fluorescens isolates. All other rRNA group I pseudomonads clustered in a manner that was in agreement with other studies, showing that the oprI gene can be useful as a complementary phylogenetic marker for classification of rRNA group I pseudomonads.Pseudomonads are increasingly being recognized as important microorganisms in our biosphere, and Pseudomonas aeruginosa and Pseudomonas fluorescens are two important representatives of this genus. As a typical opportunist, P. aeruginosa is more and more involved in a variety of often fatal nosocomial infections, in which it accounts for more than 11% of all isolates recovered (29). In cystic fibrosis, one of the most common autosomal recessive genetic diseases, it is a characteristic pathogen responsible for most of the cases of morbidity and mortality (16, 38). In general, fluorescent pseudomonads, including P. aeruginosa, Pseudomonas putida, P. fluorescens, and other species, are frequently found as rhizosphere microorganisms, in some cases promoting plant growth (11, 19, 20).P. fluorescens and P. aeruginosa are also found as inherent flora of mineral water (14, 39). Identification of fluorescent pseudomonads is often tedious and not reliable. Indeed, the present taxonomy of this group is far from clear at the finer taxonomic level, as polyphasic investigations have demonstrated (4, 13, 18, 26). Ribosomal RNAs have been applied as molecular markers with great success to unravel the rough phylogenetic structure which, at the finer level, is not always in complete agreement with the genotypic and phenotypic similarities deduced from other parts of the genome. Horizontal gene transfer, chromosomal mutation hot spots, and internal genomic rearrangements are probably the bases of these discrepancies at the species and subspecies levels. These arguments, together with the importance of discriminating phenotypic tests in routine identifications, support a polyphasic approach in bacterial taxonomy (2, 8–10, 13, 37, 40). Additional phylogenetic information requires the identification of molecules, like the recA or the gyrB genes, that are widely distributed, large enough to contain a substantial amount of information, and conserved to an appropriate degree (24, 46). In the phylogenetic tree published by Woese (43), species with the same generic name were allocated in phylogenetically distant groups. This was the case for the “genus” Pseudomonas, which is known to be a dump of assemblages of distantly related species (3, 8–10, 17). Taxonomic rearrangements of the genus Pseudomonas sensu stricto resulted in the splitting of the genus and as a logical consequence, the present genus Pseudomonas is restricted to the rRNA group I organisms, with P. aeruginosa as the type species in this group (27, 28, 37, 42, 44, 45).The oprI gene, coding for the outer membrane lipoprotein I of P. aeruginosa (5), was found to be conserved among the fluorescent pseudomonads and was considered to be a possible phylogenetic marker (6, 31). In this study, we tested whether the oprI gene could be a useful detection and identification target molecule as well as a complementary phylogenetic marker for rRNA group I pseudomonads. Also, we examined to what extent the sequence variation of the oprI gene reflects the species diversity in P. aeruginosa and P. fluorescens.     

Splitting the BLOSUM Score into Numbers of Biological Significance

Mathematical tools developed in the context of Shannon information theory were used to analyze the meaning of the BLOSUM score, which was split into three components termed as the BLOSUM spectrum (or BLOSpectrum). These relate respectively to the sequence convergence (the stochastic similarity of the two protein sequences), to the background frequency divergence (typicality of the amino acid probability distribution in each sequence), and to the target frequency divergence (compliance of the amino acid variations between the two sequences to the protein model implicit in the BLOCKS database). This treatment sharpens the protein sequence comparison, providing a rationale for the biological significance of the obtained score, and helps to identify weakly related sequences. Moreover, the BLOSpectrum can guide the choice of the most appropriate scoring matrix, tailoring it to the evolutionary divergence associated with the two sequences, or indicate if a compositionally adjusted matrix could perform better.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Proteomic Identification of Novel Secreted Antibacterial Toxins of the Serratia marcescens Type VI Secretion System

It has recently become apparent that the Type VI secretion system (T6SS) is a complex macromolecular machine used by many bacterial species to inject effector proteins into eukaryotic or bacterial cells, with significant implications for virulence and interbacterial competition. “Antibacterial” T6SSs, such as the one elaborated by the opportunistic human pathogen, Serratia marcescens, confer on the secreting bacterium the ability to rapidly and efficiently kill rival bacteria. Identification of secreted substrates of the T6SS is critical to understanding its role and ability to kill other cells, but only a limited number of effectors have been reported so far. Here we report the successful use of label-free quantitative mass spectrometry to identify at least eleven substrates of the S. marcescens T6SS, including four novel effector proteins which are distinct from other T6SS-secreted proteins reported to date. These new effectors were confirmed as antibacterial toxins and self-protecting immunity proteins able to neutralize their cognate toxins were identified. The global secretomic study also unexpectedly revealed that protein phosphorylation-based post-translational regulation of the S. marcescens T6SS differs from that of the paradigm, H1-T6SS of Pseudomonas aeruginosa. Combined phosphoproteomic and genetic analyses demonstrated that conserved PpkA-dependent threonine phosphorylation of the T6SS structural component Fha is required for T6SS activation in S. marcescens and that the phosphatase PppA can reverse this modification. However, the signal and mechanism of PpkA activation is distinct from that observed previously and does not appear to require cell–cell contact. Hence this study has not only demonstrated that new and species-specific portfolios of antibacterial effectors are secreted by the T6SS, but also shown for the first time that PpkA-dependent post-translational regulation of the T6SS is tailored to fit the needs of different bacterial species.Gram-negative bacteria have evolved several specialized protein secretion systems to secrete a wide variety of substrate proteins into the extracellular milieu or to inject them into other, often eukaryotic, cells (1). Secreted proteins and their associated secretion systems are very important in bacterial virulence and interactions with other organisms (2). One of the most recent discoveries in this field is the Type VI secretion system (T6SS),¹ which occurs widely across bacterial species (3, 4) and can target proteins to both bacterial and eukaryotic cells (5). The significance of the T6SS is becoming increasingly apparent. It has been implicated in virulence, commensalism, and symbiosis with eukaryotes (5, 6). Additionally, in many bacteria, the T6SS is now implicated in antibacterial activity. T6SS-mediated antibacterial killing appears to be important for competition between bacterial species, for example within the resident microflora of a eukaryotic host (5, 7).Secretion by the T6SS relies on 13 conserved core components which are predicted to form a large machinery associated with the cell envelope, including membrane-bound and bacteriophage tail-like subassemblies (8, 9). The membrane bound subassembly consists of inner membrane proteins (TssLM) and an outer membrane lipoprotein (TssJ) and is anchored to the cell wall. The phage tail-like assembly consists of several proteins that show structural homology with T4 phage tail proteins or are organized in similar structures (10). Hcp (TssD) proteins form hexameric rings and are thought to stack into tube-like structures (11, 12). This Hcp tube is believed to be capped by a trimer of VgrG (TssI) proteins, which share structural homology with the needle of the T4 phage tail (10, 13). In addition, VipA (TssB) and VipB (TssC) form a large tubular structure highly reminiscent of the T4 phage tail sheath (14, 15). Such similarities have led to the idea that the T6SS resembles an inverted contractile bacteriophage infection machinery and injects substrates via an Hcp/VgrG needle into other cells. Recent models propose that the VipA/B sheath surrounds the Hcp/VgrG needle and contraction of the VipA/B tube pushes the Hcp/VgrG needle out of the cell (16–18). It has been postulated that this mechanism can be triggered by close contact with other neighboring cells (19–21).Assembly, localization, and remodelling of VipA/B tubules in vivo depend on the AAA+ ATPase ClpV (TssH), another essential core component of the T6SS (14, 16, 17). ClpV also interacts with the accessory component Fha (TagH) (22, 23), which is found in a subset of T6SSs (4). The Fha protein has an N-terminal domain with a forkhead associated motif, which is predicted to bind phospho-threonine peptides (24). In Pseudomonas aeruginosa, Fha1 is phosphorylated by the Thr/Ser kinase PpkA (TagE) and dephosphorylated by the phosphatase PppA (TagG), and the phosphorylation state of Fha1 regulates the activity of the T6SS (22, 23). Phosphorylation of Fha in P. aeruginosa is also controlled by additional components, which act upstream of PpkA and form a regulatory cascade for T6SS activation (22, 25). Although homologs of PpkA and PppA have been identified in the T6SS gene clusters of certain other bacteria (3), the regulation of the T6SS by post-translational protein phosphorylation has not yet been experimentally investigated outside of Pseudomonas.To understand how the T6SS affects eukaryotic and bacterial cells, it is critical to identify substrate proteins secreted by the T6SS. The VgrG and Hcp proteins were the first identified T6SS substrates and appear to be generally secreted to the external milieu by all T6SSs (26). However, as mentioned above, Hcp and VgrG are core components of the T6SS machinery and therefore represent extracellular components of the secretion apparatus rather than genuine secreted effector proteins. Nonetheless, a limited number of VgrG homologs with extra functional effector domains at the C terminus have been identified or predicted, which account for some of the T6SS dependent effects seen against bacteria and eukaryotes. For example, the C-terminal domain of VgrG-1 from Vibrio cholerae shows actin crosslinking activity in eukaryotic cells (13, 27) and the C-terminal domain of V. cholerae VgrG-3 has bacterial cell wall hydrolase activity (28, 29).Recently, following much effort in the field, a small number of proteins secreted by the T6SS, but not structural components, have been experimentally identified. These proteins are regarded as true secreted substrates of the T6SS, with effector functions in target cells (29–35). For example, antibacterial T6SS-secreted effector proteins with peptidoglycan amidase (cell wall hydrolysis) function, the Type VI amidase effector (Tae) proteins, have been identified in Burkholderia thailandensis (32), P. aeruginosa (31), and Serratia marcescens (30). These Tae proteins play a role in T6SS-mediated antibacterial killing activity and genes encoding four families of Tae protein have been widely identified in other bacteria with T6SSs (32). T6SS-secreted effector proteins which are not peptidoglycan hydrolases have also been reported, including Tse2 secreted by P. aeruginosa, which acts in the bacterial cytoplasm (31), and the VasX and TseL proteins secreted by the V. cholerae T6SS, which are suggested to target membrane lipids (29, 34, 35). In the case of antibacterial T6SSs, the secreting bacterial cells are protected from their own T6SS effector proteins by specific immunity proteins (29–32, 35). However, given the large number of T6SSs in different bacterial species and their apparent ability to secrete multiple substrates, experimentally identified T6-secreted effector proteins still remain surprisingly scarce.Here we report the identification of multiple T6SS-secreted effector proteins in S. marcescens. S. marcescens is an opportunistic pathogen, for example causing ocular infections, nosocomial septicemia and pneumonia (36). Previously, we have identified a T6SS in S. marcescens Db10, which targets and efficiently kills other bacterial cells and plays a role in antibacterial competition (37). We have recently demonstrated that this T6SS secretes two antibacterial effectors, the Tae4 homologs Ssp1 and Ssp2, with cognate immunity proteins Rap1a and Rap2a (30).In this work, we report the analysis of the T6SS-dependent secretome of S. marcescens by label-free quantitation (LFQ) mass spectrometry and describe the identification and characterization of four novel T6SS-secreted effector proteins. These were confirmed as antibacterial toxins and specific immunity proteins were identified. Additionally, this global secretomic analysis, in combination with genetic and phosphoproteomic analyses, demonstrated that a post-translational phosphorylation system influences the ability of the S. marcescens T6SS to secrete effector proteins. Although this system uses homologs of the P. aeruginosa PpkA, PppA and Fha components, the circumstances and impact of Fha phosphorylation were shown to vary between organisms.     

Vinyl Sulfones as Antiparasitic Agents and a Structural Basis for Drug Design

Cysteine proteases of the papain superfamily are implicated in a number of cellular processes and are important virulence factors in the pathogenesis of parasitic disease. These enzymes have therefore emerged as promising targets for antiparasitic drugs. We report the crystal structures of three major parasite cysteine proteases, cruzain, falcipain-3, and the first reported structure of rhodesain, in complex with a class of potent, small molecule, cysteine protease inhibitors, the vinyl sulfones. These data, in conjunction with comparative inhibition kinetics, provide insight into the molecular mechanisms that drive cysteine protease inhibition by vinyl sulfones, the binding specificity of these important proteases and the potential of vinyl sulfones as antiparasitic drugs.Sleeping sickness (African trypanosomiasis), caused by Trypanosoma brucei, and malaria, caused by Plasmodium falciparum, are significant, parasitic diseases of sub-Saharan Africa (1). Chagas'' disease (South American trypanosomiasis), caused by Trypanosoma cruzi, affects approximately, 16–18 million people in South and Central America. For all three of these protozoan diseases, resistance and toxicity to current therapies makes treatment increasingly problematic, and thus the development of new drugs is an important priority (2–4).T. cruzi, T. brucei, and P. falciparum produce an array of potential target enzymes implicated in pathogenesis and host cell invasion, including a number of essential and closely related papain-family cysteine proteases (5, 6). Inhibitors of cruzain and rhodesain, major cathepsin L-like papain-family cysteine proteases of T. cruzi and T. brucei rhodesiense (7–10) display considerable antitrypanosomal activity (11, 12), and some classes have been shown to cure T. cruzi infection in mouse models (11, 13, 14).In P. falciparum, the papain-family cysteine proteases falcipain-2 (FP-2)⁶ and falcipain-3 (FP-3) are known to catalyze the proteolysis of host hemoglobin, a process that is essential for the development of erythrocytic parasites (15–17). Specific inhibitors, targeted to both enzymes, display antiplasmodial activity (18). However, although the abnormal phenotype of FP-2 knock-outs is “rescued” during later stages of trophozoite development (17), FP-3 has proved recalcitrant to gene knock-out (16) suggesting a critical function for this enzyme and underscoring its potential as a drug target.Sequence analyses and substrate profiling identify cruzain, rhodesain, and FP-3 as cathepsin L-like, and several studies describe classes of small molecule inhibitors that target multiple cathepsin L-like cysteine proteases, some with overlapping antiparasitic activity (19–22). Among these small molecules, vinyl sulfones have been shown to be effective inhibitors of a number of papain family-like cysteine proteases (19, 23–27). Vinyl sulfones have many desirable attributes, including selectivity for cysteine proteases over serine proteases, stable inactivation of the target enzyme, and relative inertness in the absence of the protease target active site (25). This class has also been shown to have desirable pharmacokinetic and safety profiles in rodents, dogs, and primates (28, 29). We have determined the crystal structures of cruzain, rhodesain, and FP-3 bound to vinyl sulfone inhibitors and performed inhibition kinetics for each enzyme. Our results highlight key areas of interaction between proteases and inhibitors. These results help validate the vinyl sulfones as a class of antiparasitic drugs and provide structural insights to facilitate the design or modification of other small molecule inhibitor scaffolds.