Nicholas P. Cianciotto

SUMMARY: One of the first gram-negative protein secretion systems to be discovered was the type II secretion system (T2SS), in which proteins first cross the inner membrane mainly via the Sec translocon, and then are recognized and carried across the outer membrane by the multiprotein T2SS apparatus. With its erstwhile appellation as the main terminal branch of the general secretory pathway, the T2SS has sometimes been described as (only) secreting a relatively small number of degradative enzymes for the purpose of nutrient acquisition. However, as comprehensively reviewed here, the T2SS is a great deal more than this, e.g., it (i) can secrete and expose on the cell surface over 100 proteins, (ii) is a major mediator of virulence against humans, animals, and plants, secreting diverse substrates that target essentially every step in the pathogenic process, (iii) promotes biofilm formation and environmental reactions such as metal reduction, (iv) has functional interactions with other secretion systems, and (v) has a homolog in mitochondria. Moreover, current assessment of the literature reveals an underappreciated role for the T2SS in both interbacterial antagonism and bacterial competition with fungi, protozoa, and perhaps algae. We also assembled data indicating that the T2SS mediates bacterial resistance to antibiotics. Finally, the T2SS is a fascinatingly complex “machine” whose contributing parts can vary and whose ability to recognize substrates is primarily conformational, involving spatially separated elements that interact with the T2SS apparatus through transient, multivalent interactions rather than a single dedicated binding motif.

We identified a locus within the genome of Legionella pneumophila strain 130b that encodes two related type IV pilins, PilA1 and PilA2. Based on in silico analyses, both proteins were predicted to be major pilins in all L. pneumophila strains and nearly all Legionella species. Utilizing immunoblot analysis of sheared-cell supernatants, immunofluorescence microscopy, and whole-cell ELISA, we confirmed that PilA2 (but not PilA1) behaves as a major pilin, and mutant analysis documented that PilA2-containing type IV pili (T4P) are required for DNA uptake, and subject to retraction by PilT. Unexpectedly, a pilA2 mutant, but not its complement, had impaired growth on solid and liquid media depleted of iron, indicating that PilA2 can promote growth in low-iron conditions. However, mutants lacking PilT, PilD, or PilS/PilR did not exhibit this phenotype, implying that PilA2’s newfound role does not involve inclusion into T4P, cleavage/maturation from prepilin to pilin, or the signal transduction pathway that often modulates the expression of pilin, along with other genes. Mutants lacking the FeoB Fe ²⁺ transporter, LbtC siderophore transporter, or Ccm cytochrome maturation showed even greater defects on low-iron agar media when pilA2 mutations were introduced into them, suggesting that PilA2 functions within a novel iron-uptake pathway. Compatible with this, the pilA2 mutant, but not its complement, exhibited increased resistance to streptonigrin and heightened expression of iron-regulated genes. Finally, although unimpaired for growth within amoebae, the pilA2 mutant displayed increased biofilm formation. Overall, this study reveals a novel role for a type IV pilin in bacterial growth in iron acquisition/assimilation. IMPORTANCE: Legionella pneumophila is the main cause of Legionnaires’ disease, an increasingly prevalent pneumonia. Prior studies indicated that L. pneumophila expresses type IV pili, a common form of pili that is mainly known for mediating DNA uptake, twitching motility, and attachment to biotic and abiotic surfaces. By utilizing a variety of in silico and experimental approaches, wehave helped redefine the major structural component (pilin) of the L. pneumophila pili. More significantly, we uncovered a heretofore unknown and seemingly novel role for the pilin in L. pneumophila growth in low-iron conditions and iron acquisition/assimilation. These findings have broad implications for understanding other bacteria and infectious diseases, as well as offering fresh insight into the roles of pilins in bacterial physiology and the mechanisms of iron assimilation.

Previously, it was demonstrated that a chitinase (ChiA) secreted by the Legionella pneumophila type II secretion system (T2SS) degrades mucin, thereby facilitating bacterial movement through a mucin layer. Here, we discovered that the addition of mucin to a chemically defined medium (CDM) greatly stimulates the growth of L. pneumophila , indicating that the bacterium can very effectively utilize mucin as a food source. This growth-stimulatory effect was evident in broth and agar media and for all WT strains tested. Remarkably, the growth of L. pneumophila on mucin-containing CDM agar rivalled the growth of the bacterium on buffered charcoal yeast extract (BCYE) agar, the standard medium used for cultivating legionellae. A L. pneumophila mutant lacking the T2SS was majorly impaired for growth in mucin-containing CDM, suggesting that exoenzymes are needed for mucin assimilation. In support of this hypothesis, mutants lacking either ChiA or the secreted protease ProA were also impaired for growth in the presence of added mucin. Finally, we observed that L. pneumophila , but not a mutant lacking secreted surfactant, exhibits a marked spreading phenotype (i.e. sliding motility) when grown on CDM agar vs. BCYE agar. However, WT bacteria grown on CDM agar containing mucin did not show this spreading, suggesting that sliding motility is induced under low-nutrient conditions but repressed by mucin byproducts. Taken together, these results provide new insight into the versatility of L. pneumophila physiology and suggest that L. pneumophila may grow well in extracellular spaces in the lungs that contain mucin.

Prior analyses suggested that the type II secretion system (T2SS) of Legionella pneumophila secretes ≥47 proteins beyond its 26 known substrates. Upon examination of mutants of wild-type strain 130b that lack those exoproteins most conserved across the Legionella genus, we discovered that protein “06635” majorly promotes L. pneumophila replication within amoebae. Immunoblotting, proteomics, and whole-cell enzyme-linked immunosorbent assay (ELISA) confirmed that 06635 exists in culture supernatants and on the bacterial outer surface and does so in a T2SS-dependent manner. Bioinformatic analyses identified 06635 as a novel member of the carbohydrate esterase family 4, whose members deacetylate bacterial surface polysaccharides, peptidoglycan, chitins, and/or xylans. Given 06635’s T2SS-dependent secretion, low-level amino acid similarity to known peptidoglycan deacetylases, and the unaltered lysozyme resistance of a 06635 mutant, we pursued the hypothesis that 06635 deacetylates a polysaccharide on L. pneumophila ’s surface. Supporting this, the 06635 mutant exhibited increased binding to both wheat germ agglutinin (i.e., more surface N -acetylglucosamine) and antibodies that recognize acetylated lipopolysaccharide (LPS). Nuclear magnetic resonance (NMR) analysis of isolated mutant vs wild-type LPS confirmed that 06635 promotes LPS deacetylation. Thus, we designated 06635 as PdaA, for p olysaccharide d e a cetylase A . Compatible with its altered surface, the pdaA mutant showed greater autoaggregation, increased biofilm formation, and heightened sensitivity to both polymyxin and human serum. Thus, we hypothesize that, following its secretion via the T2SS, PdaA deacetylates LPS, and perhaps other moieties, impacting many significant processes. While defining PdaA, we identified many more putative substrates of the L. pneumophila T2SS, bringing the size of the T2SS output to approximately 120. IMPORTANCE: Legionella pneumophila is the principal cause of Legionnaires’ disease, an increasingly common form of pneumonia. Although prior work demonstrated that the bacterium utilizes its type II protein secretion system (T2SS) to survive in aquatic environments and to cause lung infection, the full scope and impact of this Legionella secretion system is still relatively underappreciated. By utilizing an expanded proteomic approach and testing newly made mutants in a wide range of assays, we have determined that the L. pneumophila type II secretome encompasses approximately 120 proteins, and among these proteins is a novel polysaccharide deacetylase (PdaA) that modulates the L. pneumophila surface and lipopolysaccharide, impacting intracellular infection, biofilm formation, and resistance to both antibiotics and human serum. Moreover, since T2SSs and homologs of PdaA were found in many other bacteria, our findings should also have implications for understanding other infectious diseases and environmental processes.

We report the draft genome sequences of Stenotrophomonas maltophilia strains UPSm1, UPSm3, and UPSm5 and Stenotrophomonas pavanii strain UPSm2, all of which had been isolated from the respiratory tract of human patients. The genome assemblies contain single contigs that have an average length of 4,864,360 bp.

Clinical and environmental isolates of Stenotrophomonas maltophilia produce an enterobactin-like siderophore that promotes bacterial growth under low-iron conditions. Although prior mutational and bioinformatic analyses indicated that most of the enzymes encoded by the S. maltophilia entCEBB′FA locus are suitably reminiscent of their counterparts in Escherichia coli and other bacteria, Stenotrophomonas EntB was unusual. In bacteria producing enterobactin-related molecules, EntB and its homologs are usually multi-domain proteins in which the amino portion acts as an isochorismatase and the carboxy domain serves as an aryl carrier protein (ArCP). However, in S. maltophilia the isochorismatase and ArCP functions are encoded by two distinct genes: entB and entB′ , respectively. Current mutant analysis was used to first confirm that S. maltophilia entB is needed for siderophore activity and bacterial growth in iron-depleted media. A crystal structure of S. maltophilia EntB was then obtained. The structure aligned with the N-terminal portion of EntB from E. coli and VibB from Vibrio cholerae , affirming the protein to be a single-domain isochorismatase. However, S. maltophilia EntB also aligned with the single-domain PhzD from Pseudomonas aeruginosa , which is a key enzyme involved in the biosynthesis of the antimicrobial compound phenazine. BLASTP searches indicated that entB and its neighboring genes are fully conserved amongst S. maltophilia strains but are variably present in other Stenotrophomonas species. The closest homologs to S. maltophilia EntB outside the genus were hypothetical proteins/putative isochorismatases in some Gram-negative bacteria (for example Pseudomonas spp. and Xanthomonas spp.), Gram-positive bacteria ( Streptomyces spp. and Bacillus subtilis ) and fungi (for example Rhizopus arrhizus and Knufia peltigerae ).

To discover new factors that are involved in iron acquisition by Legionella pneumophila , we used RNA-Seq to identify the genes that are most highly induced when virulent strain 130b is cultured in a low-iron chemically defined medium. Among other things, this revealed 14915 , a heretofore uncharacterized gene that is predicted to be transcriptionally regulated by Fur and to encode a novel, ~15 kDa protein. 14915 was present in all L. pneumophila strains examined and had homologs in a subset of the other Legionella species. Compatible with it containing a classic signal sequence, the 14915 protein was detected in bacterial culture supernatants in a manner dependent upon the L. pneumophila type II secretion system. Thus, we designated 14915 as IrsA for ir on- r egulated, s ecreted protein A . Based on mutant analysis, the irsA gene was not required for optimal growth of strain 130b in low-iron media. However, after discovering that the commonly used laboratory-derived strain Lp02 has a much greater requirement for iron, we uncovered a growth-enhancing role for IrsA after examining an Lp02 mutant that lacked both IrsA and the Fe ²⁺ -transporter FeoB. The irsA mutant of 130b, but not its complemented derivative, did, however, display increased biofilm formation on both plastic and agar surfaces, and compatible with this, the mutant hyper-aggregated. Thus, IrsA is a novel, iron-regulated exoprotein that modulates biofilm formation and, under some circumstances, promotes growth in low-iron conditions. For this study, we determined and deposited in the database a complete and fully assembled genome sequence for strain 130b. IMPORTANCE: The bacterium Legionella pneumophila is the principal cause of Legionnaires’ disease, a potentially fatal form of pneumonia that is increasing in incidence. L. pneumophila exists in many natural and human-made water systems and can be transmitted to humans through inhalation of contaminated water droplets. L. pneumophila flourishes within its habitats by spreading planktonically, assembling into biofilms, and growing in larger host cells. Iron acquisition is a key determinant for L. pneumophila persistence in water and during infection. We previously demonstrated that L. pneumophila assimilates iron both by secreting a non-protein iron chelator (siderophore) and by importing iron through membrane transporters. In this study, we uncovered a novel, secreted protein that is highly iron-regulated, promotes L. pneumophila ’s growth in low-iron media, and impacts biofilm formation. We also identified uncharacterized, IrsA-related proteins in other important human and animal pathogens. Thus, our results have important implications for understanding iron assimilation, biofilm formation, and pathogenesis.

Interbacterial antagonism involves all major phyla, occurs across the full range of ecological niches, and has great significance for the environment, clinical arena, and agricultural and industrial sectors. Though the earliest insight into interbacterial antagonism traces back to the discovery of antibiotics, a paradigm shift happened when it was learned that protein secretion systems (e.g., types VI and IV secretion systems) deliver toxic “effectors” against competitors. However, a link between interbacterial antagonism and the Gram-negative type II secretion system (T2SS), which exists in many pathogens and environmental species, is not evident in prior reviews on bacterial competition or T2SS function. A current examination of the literature revealed four examples of a T2SS or one of its known substrates having a bactericidal activity against a Gram-positive target or another Gram-negative. When further studied, the T2SS effectors proved to be peptidases that target the peptidoglycan of the competitor. There are also reports of various bacteriolytic enzymes occurring in the culture supernatants of some other Gram-negative species, and a link between these bactericidal activities and T2SS is suggested. Thus, a T2SS can be a mediator of interbacterial antagonism, and it is possible that many T2SSs have antibacterial outputs. Yet, at present, the T2SS remains relatively understudied for its role in interbacterial competition. Arguably, there is a need to analyze the T2SSs of a broader range of species for their role in interbacterial antagonism. Such investigation offers, among other things, a possible pathway toward developing new antimicrobials for treating disease.

Stenotrophomonas maltophilia expresses a type IV protein secretion system (T4SS) that promotes contact-dependent killing of other bacteria and does so partly by secreting the effector TfcB. Here, we report the structure of TfcB, comprising an N-terminal domain similar to the catalytic domain of glycosyl hydrolase (GH-19) chitinases and a C-terminal domain for recognition and translocation by the T4SS. Utilizing a two-hybrid assay to measure effector interactions with the T4SS coupling protein VirD4, we documented the existence of five more T4SS substrates. One of these was protein 20845, an annotated nuclease. A S. maltophilia mutant lacking the gene for 20845 was impaired for killing Escherichia coli , Klebsiella pneumoniae , and Pseudomonas aeruginosa . Moreover, the cloned 20845 gene conferred robust toxicity, with the recombinant E. coli being rescued when 20845 was co-expressed with its cognate immunity protein. The 20845 effector was an 899 amino-acid protein, comprised of a GHH-nuclease domain in its N-terminus, a large central region of indeterminant function, and a C-terminus for secretion. Engineered variants of the 20845 gene that had mutations in the predicted catalytic site did not impede E. coli , indicating that the antibacterial effect of 20845 involves its nuclease activity. Using flow cytometry with DNA staining, we determined that 20845, but not its mutant variants, confers a loss in DNA content of target bacteria. Database searches revealed that uncharacterized homologs of 20845 occur within a range of bacteria. These data indicate that the S. maltophilia T4SS promotes interbacterial competition through the action of multiple toxic effectors, including a potent, novel DNase. IMPORTANCE: Stenotrophomonas maltophilia is a multi-drug-resistant, Gram-negative bacterium that is an emerging pathogen of humans. Patients with cystic fibrosis are particularly susceptible to S. maltophilia infection. In hospital water systems and various types of infections, S. maltophilia co-exists with other bacteria, including other pathogens such as Pseudomonas aeruginosa . We previously demonstrated that S. maltophilia has a functional VirB/D4 type VI protein secretion system (T4SS) that promotes contact-dependent killing of other bacteria. Since most work on antibacterial systems involves the type VI secretion system, this observation remains noteworthy. Moreover, S. maltophilia currently stands alone as a model for a human pathogen expressing an antibacterial T4SS. Using biochemical, genetic, and cell biological approaches, we now report both the discovery of a novel antibacterial nuclease (TfdA) and the first structural determination of a bactericidal T4SS effector (TfcB).