Expanding Role of Type II Secretion in Bacterial Pathogenesis and Beyond

ABSTRACT

Type II secretion (T2S) is one means by which Gram-negative pathogens secrete proteins into the extracellular milieu and/or host organisms. Based upon recent genome sequencing, it is clear that T2S is largely restricted to the Proteobacteria, occurring in many, but not all, genera in the Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Deltaproteobacteria classes. Prominent human and/or animal pathogens that express a T2S system(s) include Acinetobacter baumannii, Burkholderia pseudomallei, Chlamydia trachomatis, Escherichia coli, Klebsiella pneumoniae, Legionella pneumophila, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Vibrio cholerae, and Yersinia enterocolitica. T2S-expressing plant pathogens include Dickeya dadantii, Erwinia amylovora, Pectobacterium carotovorum, Ralstonia solanacearum, Xanthomonas campestris, Xanthomonas oryzae, and Xylella fastidiosa. T2S also occurs in nonpathogenic bacteria, facilitating symbioses, among other things. The output of a T2S system can range from only one to dozens of secreted proteins, encompassing a diverse array of toxins, degradative enzymes, and other effectors, including novel proteins. Pathogenic processes mediated by T2S include the death of host cells, degradation of tissue, suppression of innate immunity, adherence to host surfaces, biofilm formation, invasion into and growth within host cells, nutrient assimilation, and alterations in host ion flux. The reach of T2S is perhaps best illustrated by those bacteria that clearly use it for both environmental survival and virulence; e.g., L. pneumophila employs T2S for infection of amoebae, growth within lung cells, dampening of cytokines, and tissue destruction. This minireview provides an update on the types of bacteria that have T2S, the kinds of proteins that are secreted via T2S, and how T2S substrates promote infection.

INTRODUCTION

Secreted proteins have a major role in the pathogenesis of bacterial infections, including important diseases of humans, animals, and plants. In the case of Gram-negative bacteria, there are seven secretion systems (types I, II, III, IV, V, VI, and IX) that mediate the export of “effector” proteins out of the bacterial cell and into the extracellular milieu or into target host cells (1–3). Type II secretion (T2S) was the first such system to be defined, based upon work done in the mid-1980s on pullulanase secretion by Klebsiella oxytoca (4). Further insight into T2S was then gained from the examination of Aeromonas, Pseudomonas, Vibrio, and a few additional members of the gammaproteobacteria (5, 6). Thus, T2S is considered a two-step process; i.e., proteins to be secreted are first carried across the inner membrane (IM) and into the periplasm by the Sec translocon (7) or Tat pathway (8) and then, after folding into a tertiary conformation (and in some instances, undergoing oligomerization), are transported across the outer membrane (OM) by the dedicated T2S apparatus (2). The T2S machinery is made up of 12 “core” proteins, which are denoted here as T2S C, D, E, F, G, H, I, J, K, L, M, and O (9, 10). In recent years, there has been remarkable progress toward elucidating the precise structure of the T2S apparatus (2, 11–16). In essence, there are four subcomplexes: (i) an OM “secretin,” which is a pentadecamer of the T2S D protein that provides a pore through the membrane; (ii) an IM platform composed of T2S C, F, L, and M, with T2S C providing a connection to the OM secretin; (iii) a cytoplasmic ATPase, which is a hexamer of T2S E that is recruited to the IM platform; and (iv) a periplasm-spanning pseudopilus which is a helical filament of the major pseudopilin T2S G capped by the minor pseudopilins T2S H, I, J, and K. Finally, T2S O is an IM peptidase that cleaves and methylates the pseudopilins as a prelude to their incorporation into the pseudopilus. Thus, during T2S, protein substrates present in the periplasm are delivered to the T2S apparatus, presumably following their recognition by T2S C and T2S D (17), and then using energy generated at the IM, the pseudopilus acts as a piston or an Archimedes screw to push the proteins through the OM secretin (2). Although recent papers have detailed the structure of the T2S apparatus and the molecular mechanism of secretion (11–15), it has been some time since there was a review focused on the prevalence of T2S and its role in pathogenesis. Hence, this minireview will provide an update on the types of bacteria and pathogens that have T2S, the numbers and kinds of proteins that are secreted via T2S, and how T2S-dependent proteins promote infection.

Prevalence of type II protein secretion systems.

Following the advent of whole-genome sequencing, complete or nearly complete sets of T2S genes (i.e., containing all or almost all of the core constituents, T2S CDEFGHIJKLMO) were identified in 32 genera of Proteobacteria, comprising 22 genera in the gammaproteobacteria, 4 genera each in the alpha- and betaproteobacteria, and 2 genera in the deltaproteobacteria (10, 18). However, T2S genes were absent from 29 other genera of Proteobacteria, including those in the epsilonproteobacteria, indicating that T2S occurs in many, but not all, genera in the phylum Proteobacteria (10). Extending this analysis, a recent study, which defined the full set of T2S genes as one encoding T2S CDEFGHIJKLMNO, identified the system in 360 of the 1,528 Gram-negative genomes examined, with 58%, 45%, 15%, 6%, and 0% prevalence among beta-, gamma-, delta-, alpha-, and epsilonproteobacteria, respectively (19). Figure 1 depicts the distribution of T2S within the evolutionary tree of the Proteobacteria. Looking beyond the Proteobacteria, there are interesting examples of organisms that have a smaller number of T2S-related genes; e.g., Leptospira interrogans of the Spirochaetes encodes T2S CDEFGJKLMO, Chlamydia and Chlamydophila species within the Chlamydiae harbor genes for T2S CDEFG, Rhodopirellula baltica belonging to the Planctomycetes may encode T2S DEFGIKO, Aquifex aeolicus of the Aquificae carries homologs for T2S DEFGO, and Thermotoga maritima of the Thermotogae appears to encode T2S DEFG (10, 19–22). In the case of Chlamydia trachomatis, one of these genes has been linked to protein secretion (20), suggesting that there may be different subclasses of T2S that deviate from the canonical system present in the Proteobacteria. In Synechococcus elongatus belonging to the Cyanobacteria, a T2S E-like gene has been linked to protein secretion; however, this gene may be encoding a component of a type IV pilus rather than a T2S apparatus (19, 23). So far, genome database analyses have failed to reveal any evidence for potential T2S systems in Bacteroidetes, Chlorobi, Fusobacteria, or Verrucomicrobia (10, 19). Thus, despite the fact that T2S is often referred to as the main terminal branch of the general secretory pathway (5, 24, 25), T2S is not, by any means, conserved among Gram-negative (“diderm”) bacteria. Rather, in its canonical form, T2S is largely restricted to the Proteobacteria (Fig. 1). Furthermore, even in the Proteobacteria, T2S, though common, is not universal. Put another way, T2S may be no more prevalent across Gram-negative genera than is type I, III, IV, V, or VI secretion (19). Hence, T2S is best considered a specialized secretion system that a subset of Gram-negative bacteria has evolved to utilize for their growth within the environment or larger hosts. Table 1 shows a comprehensive list of those bacteria in which T2S has been shown by mutational analysis to actually be functional.

FIG 1.

Representative distribution of T2S genes among the Proteobacteria. An unrooted phylogenetic tree of the Proteobacteria and several other bacteria was constructed with aligned 16S rRNA sequences (65, 66) using standard neighbor-joining methods (67, 68). Genus names are denoted at each leaf. Clades representing the alpha-, beta-, gamma-, delta-, and epsilonproteobacteria are identified by the α, β, γ, δ, and ε Greek symbols. The bar represents the number of nucleotide substitutions per site. Bacteria that have been demonstrated to express a functional T2S system are indicated in green. Representative bacteria that have a complete or nearly complete set of T2S genes but for which functionality has not yet been shown are indicated in red. Representative bacteria that lack T2S genes are indicated in black.

TABLE 1.

Secreted proteins, activities, and phenotypes dependent upon T2S^a

Category and bacterium	Frequent niche/pathogenicityb	Secreted protein(s)/activity(ies)	Phenotype(s)	Reference(s)
Human and/or animal pathogens (including some rare pathogens, as indicated)
Acinetobacter baumannii	Soil, water/pathogen of humans (e.g., pneumonia)	Lipase LipA, phospholipase LipAN, and >10 other secreted proteins identified by proteomics	Survival in neutropenic mouse model, growth in murine pneumonia model	69, 70
Acinetobacter calcoaceticus	Soil, intestinal tract/pathogen of humans (rare)	Esterase, lipase	Dodecane degradation	71
Acinetobacter nosocomialis	Soil/pathogen of humans (rare)	Lipases LipA and LipH, protease CpaA, and 57 other secreted proteins identified by proteomics		72
Aeromonas hydrophila	Freshwater or brackish water/pathogen of humans (e.g., septicemia) amphibians, and fish	Aerolysin (Act enterotoxin), amylase, DNase, glycerophospholipid cholesterol acyltransferase, protease, S-protein	Cytotoxicity, inflammatory signaling from macrophages and epithelial cells, virulence in mice	6, 73–78
Aeromonas salmonicida	Freshwater/pathogen of fish (e.g., salmon)	Aerolysin		79
Burkholderia cenocepacia	Soil, water/pathogen of humans (lung infection)	Lipase, polygalacturonase, proteases ZmpA and ZmpB	Cleavage of host tissue and defense proteins, virulence in rat lung infection, pathology in Caenorhabditis elegans infection model, virulence in onion infection, survival in macrophages, triggering IL-1β secretion from macrophages	80–83
Burkholderia pseudomallei	Soil/pathogen of animals and humans (mellioidosis)	Chitinase, deubiquitinase TssM, lipase, phospholipases C, proteases, and ∼40 other proteins identified by proteomics	Virulence in a hamster model, suppression of innate immune response	28, 84, 85
Burkholderia vietnamiensis	Soil/pathogen of humans (lung infection)	Hemolysin, phospholipase C		86
Chlamydia trachomatis	Human genital tract/pathogen of humans (STD, conjunctivitis)	CPAF serine protease, putative glycogen hydrolase	Intracellular growth in epithelial cells, generation of infectious EBs, cleavage of vimentin, lamin-associated protein 1, and host antimicrobial peptides	20, 87–89
Escherichia coli
Enteroaggregative	Intestinal tract/pathogen of humans (diarrhea)	Mucin-degrading metalloprotease YghJ	Degradation of mucous layer	90
Enterohemorrhagic	Intestinal tract/pathogen of humans (diarrhea, hemolytic-uremic syndrome)	Metalloprotease StcE, protein YodA	Cleaves C1 esterase inhibitor and mucin 7, adherence to epithelia, colonization of the intestine in a rabbit model	91–93
Enteropathogenic	Intestinal tract/pathogen of humans (diarrhea)	Outer membrane and secreted forms of lipoprotein SslE	Biofilm formation, virulence in a rabbit model of disease	94, 95
Enterotoxigenic	Intestinal tract/pathogen of humans (diarrhea, cholera-like)	For the beta system, heat-labile (LT) toxin, mucin-degrading protease YghJ, various membrane proteins	Degradation of mucous layer, diarrhea	90, 96–98
		For the alpha system, none identified		99
Extraintestinal	Intestinal tract/pathogen of humans (meningitis)	Putative lipoprotein ECOK1_3385	Protective antigen in murine sepsis model	100
Uropathogenic	Intestinal and urinary tracts/pathogen of humans (UTI)	Surface-expressed DraD invasin	Adherence to epithelial cells, survival in murine model of UTI	101, 102
Klebsiella oxytoca	Soil, freshwater, plants/pathogen of humans (rare)	Surface-associated pullulanase		103
Klebsiella pneumoniae	Soil, water, intestinal tract/pathogen of humans (pneumonia)	Pullulanase	Blockade of NF-κB in epithelial cells, growth in the murine lung	104
Legionella pneumophila	Freshwater, soil/pathogen of humans (pneumonia)	Aminopeptidases LapA and LapB; chitinase ChiA; collagen-like protein Lcl; diacylglycerol lipase; endoglucanase CelA; glucoamylase GamA; glycerophospholipid cholesterol acyltransferase PlaC; lysophospholipase A PlaA; metalloprotease ProA; mono- and triacylglycerol lipase LipA; six novel proteins including NttA, NttB, and NttC; phospholipases C PlcA and PlcB; putative amidase Lpg0264; putative astacin-like peptidase LegP; putative peptidyl-proline cis-trans-isomerase; RNase SrnA; tartrate-resistant acid phosphatase; tartrate-sensitive acid phosphatase Map; triacylglycerol lipase LipB; VirK-like protein Lpg1832	Virulence in murine pneumonia model (bacterial growth and tissue damage), disruption of PMN function, intracellular growth in macrophages and epithelial cells, recruitment of host GTPase Rab1B to the Legionella-containing vacuole, dampening of cytokine production by host cells, intracellular growth in environmental amoebae, colony morphology and autoaggregation, growth at low temperatures, poly-3-hydroxybutyrate storage, biofilm formation, sliding motility and surfactant production	26, 46–50, 53–56, 58–61, 105–111
Photobacterium damselae	Marine water/pathogen of fish and humans (rare)	Phospholipase D Dly, pore-forming toxins HlyApl and HlyAch	Virulence in marine fish	112
Pseudomonas aeruginosa	Soil, plants, water/pathogen of humans (pneumonia)	For the Xcp system, alkaline phosphatase PhoA; aminopeptidase PaAP; chitin-binding protein CbpD; DNase PA3909; elastases LasA and LasB; exotoxin A; glycerophosphoryl diester phosphatase GlpQ; lipases LipA and LipC; lipoxygenase LoxA; mucinases; novel proteins PA2377 and PA4140; phosphodiesterase PA3910; phospholipases C PlcH (hemolytic), PlcN, and PlcB; protease IV; putative protease PmpA	Cytotoxicity, degradation of host tissue (e.g., in lung and eye) and surfactant proteins, disruption of cell-cell junctions (e.g., cadherin cleavage), virulence in a murine lung infection model, regulation of siderophore (pyoverdine) expression, swarming motility	113–122
		For the Hxc system, phosphatase LapA; DING homolog LapC; surface-expressed PstS	Adherence to epithelial cells, survival in low-phosphate conditions	116, 123, 124
		For the Txc system, chitin-binding protein CbpE		125
Pseudomonas alcaligenes	Soil/pathogen of humans (rare)	Lipase LipA		126
Stenotrophomonas maltophilia	Soil, water, plants/pathogen of humans (pneumonia, bacteremia)	For the Xps system, proteases StmPr1 and StmPr2 and >5 secreted proteins by SDS-PAGE	Detachment and cytotoxicity toward epithelial cells, degradation of extracellular matrix and cytokines	127, 128
		For the Gsp system, none identified yet		127, 128
Vibrio anguillarum	Marine water/pathogen of fish and eels	Metalloprotease EmpA		129
Vibrio cholerae	Marine water, shellfish/pathogen of humans (cholera)	Aminopeptidases Lap and LapX; biofilm matrix proteins RbmA, RbmC, and Bap1; chitin-degrading enzymes including chitinase ChiA-1; chitin-binding protein GbpA; cholera toxin; collagenase VchC; cytolysin VCC; hemagglutinin-protease HapA; lipase; neuraminidase; serine proteases VesA, VesB, and VesC; TagA-related protein; and three novel proteins identified by proteomics	Watery diarrhea, degradation of mucous layers, attachment to epithelial cells, attachment to biotic (e.g., copepods) and abiotic surfaces in aquatic environments, biofilm formation, degradation of the matrix that covers the eggs of chironomids	34, 35, 39, 41, 130–133
Vibrio parahaemolyticus	Marine water, shellfish/pathogen of humans (gastroenteritis)	Lipase		134
Vibrio vulnificus	Marine water, shellfish/pathogen of humans (septicemia)	Chitinase; cytolysin; elastase VvpE; hemolysin VvhA; putative peptidyl-proline cis-trans-isomerase; proteases including serine protease VvpS, sugar hydrolase, and several outer membrane proteins	Cytotoxicity, virulence in murine models	135–137
Yersinia enterocolitica	Intestinal tracts of mammals and various other animals/pathogen of animals and humans (gastroenteritis)	For the Yts1 system, GlcNAc-binding proteins ChiY and EngY, YE3650	Virulence in murine model	138, 139
		For the Yts2 system, none identified yet	Intracellular infection of macrophages	138–140
Plant pathogens (including, as indicated, some that afflict humans on rare occasions)
Burkholderia gladioli	Soil, water/pathogen of various plants and fungi and humans (rare)	Chitinase, protease	Cavity disease in mushrooms	141
Burkholderia glumae	Soil/pathogen of rice plants	Lipase LipA, proteases, 32 other proteins identified by proteomic analysis	Virulence in rice infection	142, 143
Dickeya dadantii (formerly Erwinia chrysanthemi)	Soil, water/pathogen of various vegetables and flowers (soft rot)	For the Out system, Avr-like protein AvrL; cellulase Cel5; esterase FaeD; pectate lyases PelA, PelB, PelC, PelD, PelE, PelI, PelL, PelN, and PelZ; pectin acetylesterase PaeY; pectin methylesterase PemA; rhamnogalacturonan lyase RhiE	Soft-rot disease in plants, activation of plant innate immune system, growth promotion of EHEC on lettuce	144–149
		For the Stt system, outer membrane-anchored PnlH		150
Erwinia amylovora	Soil/pathogen of fruit (fire blight)	Levansucrase, polygalacturonase	Infection of pear tissue, low-temperature growth	151–153
Pectobacterium carotovorum (formerly Erwinia carotovora)	Soil, water/pathogen of many plants (soft rot)	Cellulases CelV and CelB; necrosis-inducing protein (Nip); pectin lyases PelA, PelB, PelC, PelZ, Pel-3, and ECA2553; novel secreted proteins ECA2134, ECA3580, and ECA3946; polygalacturonases PehA and PehX; putative cellulase ECA2220; putative proteoglycan hydrolase ECA0852; putative virulence protein Svx	Virulence in plant model, maceration of tobacco leaf	154–157
Pectobacterium wasabiae	Soil/pathogen of plants (wasabi)	Necrosis-inducing protein (Nip)	Soft-rot disease in tubers	158
Ralstonia solanacearum	Soil/pathogen of many types of plants (wilt disease)	Cellulase, pectin methylesterase, cellobiosidase, polygalacturonases, >30 proteins identified by proteomics	Colonization of plants, causing wilting disease	159, 160
Xanthomonas axonopodis	Soil, freshwater/pathogen of citrus plants (citrus canker)	Cellulase, polygalacturonases, proteases	Virulence in orange leaves	161, 162
Xanthomonas campestris pv. campestris	Soil, freshwater/pathogen of crucifer plants (black rot)	α-Amylase; cellulase; pectate lyase; polygalacturonases PghAxc and PghBxc; serine protease PrtA; other proteases	Virulence in Arabidopsis plant models	163–165
Xanthomonas campestris pv. vesicatoria	Soil, freshwater/pathogen of pepper and tomato plants (leaf spot)	For the Xps system, lipase XCV0536, protease XCV3671, and xylanases XynC, XCV4358, and XCV4360	Virulence in pepper plant model	166, 167
		For the Xcs system, none identified yet		166
Xanthomonas oryzae pv. oryzae	Soil, freshwater/pathogen of various rice plants (rice blight)	Cellulase ClsA, cysteine protease CysP2, endoglucanase EglXoB, lipase LipA, polysaccharide, putative cellobiosidase CbsA, xylanase XynB	Virulence in rice infection model	168–173
Xanthomonas oryzae pv. oryzicola	Soil, freshwater/pathogen of various rice plants (rice blight)	Cysteine protease XOC1601, polygalacturonase XOC2128, protease EcpA, protease XOC3806	Virulence in rice infection model	174, 175
Xylella fastidiosa	Plant xylem/pathogen of various plants (e.g., grapes)	Lipase/esterase LesA	Virulence in grapevine infection	176
Nonpathogens or, as indicated, bacteria that very rarely cause human disease
Aeromonas veronii	Freshwater, leech symbiont/pathogen of humans (very rare)	Hemolysin	Colonization of the medicinal leech (symbiosis)	177
Caulobacter crescentus	Freshwater/nonpathogen	Outer membrane and secreted lipoprotein ElpS	Activates alkaline phosphatase activity	178
Cellvibrio japonicus	Soil/nonpathogen	Endoglucanase		179
Cupriavidus metallidurans	Soil/nonpathogen	Alkaline phosphatase		180
Nonpathogenic Escherichia coli	Intestinal tract/nonpathogen	Chitinase, lipoprotein SslE		181, 182
Geobacter sulfurreducens	Sediments/nonpathogen	Multi-copper oxidase OmpB		183
Gluconacetobacter diazotrophicus	Symbiont of sugar cane and other plants/nonpathogen	Levansucrase		184
Marinobacter hydrocarbonoclasticus	Marine water/nonpathogen	Lipase	Biofilm formation	185
Methylococcus capsulatus	Fresh and marine water, sediment/nonpathogen	Serine protease MCA0875, surface-associated protein MCA2589, c-type cytochrome MCA0338		186
Pseudoalteromonas haloplanktis	Marine water/nonpathogen	Protease		187
Pseudoalteromonas ruthenica	Marine water/nonpathogen	CPI protease		188
Pseudoalteromonas tunicata	Marine water/nonpathogen	>30 proteins identified by proteomics	Iron acquisition, pigmentation	189
Pseudomonas fluorescens	Soil, plants, water/nonpathogen	DING homolog Psp		190
Pseudomonas putida	Soil/nonpathogen	For the Xcp system, surface-expressed phosphatase UxpB	Growth in low-phosphate media	191
		For the Xcm system, surface-expressed Mn-oxidizing enzyme(s)		192
Ralstonia pickettii	Soil, freshwater/primarily nonpathogen; pathogen of humans (very rare)	Poly(3-hydroxybutyrate) depolymerase		193
Shewanella oneidensis	Water/primarily nonpathogen; pathogen of humans (very rare)	Outer membrane proteins, including c-type cytochromes MtrC and OmcA, and DMSO reductase DmsA	Fe(III) and Mn(IV) reduction, extracellular respiration	194–197
Vibrio fischeri (now Allivibrio fischeri)	Marine water, symbiont of squid/nonpathogen	NAD+-glycohydrolases HvnA and HvnB		18, 198

^aDependence is based upon the absence or reduction of the indicated protein, activity, or phenotype in a mutant(s) specifically lacking a T2S gene(s). Abbreviations: IL-1β, interleukin 1β; STD, sexually transmitted disease; CPAF, chlamydial protease-like activity factor; EBs, elementary bodies; UTI, urinary tract infection; PMN, polymorphonuclear leukocytes; EHEC, enterohemorrhagic E. coli; CPI, cysteine protease inhibitor; DMSO, dimethyl sulfoxide.

^bOnly the most common disease manifestation(s) is noted.

T2S in pathogens of humans and animals.

The human pathogens that are known to possess functional T2S include representatives from 10 genera of gammaproteobacteria (Acinetobacter, Aeromonas, Escherichia, Klebsiella, Legionella, Photobacterium, Pseudomonas, Stenotrophomonas, Vibrio, and Yersinia), one genus of betaproteobacteria (Burkholderia), and one genus of Chlamydiae (Chlamydia) (Fig. 1). Among the prominent human pathogens that use T2S are Acinetobacter baumannii, Aeromonas hydrophila, Burkholderia cenocepacia, Burkholderia pseudomallei, Chlamydia trachomatis, Escherichia coli, Klebsiella pneumoniae, Legionella pneumophila, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, and Yersinia enterocolitica (Table 1). Some of these T2S-expressing bacteria are also natural pathogens of animals, ranging from those afflicting fish (e.g., A. hydrophila, Aeromonas salmonicida, Photobacterium damselae, and V. anguillarum) to those impacting other mammals (e.g., B. pseudomallei and Y. enterocolitica). Based upon genome sequencing and Southern blot analyses, it is likely that additional pathogenic members of these genera employ T2S (10, 26–28). In most cases, these human and animal pathogens encode a single T2S system. Yet, for some strains of E. coli, P. aeruginosa, S. maltophilia, and Y. enterocolitica, there are two or three distinct T2S systems (Table 1). As more isolates are sequenced, there will likely be additional examples of multiple sets of T2S genes. At present, the functionality of the second system in E. coli, S. maltophilia, and Y. enterocolitica is unknown, as no secreted substrates or activities have been defined. A specialized growth condition(s) may be needed in order for the expression of a T2S system to be evident; e.g., whereas expression of the Xcp T2S system of P. aeruginosa is easily observed in bacteriological media, expression of the Hxc system occurs only in low phosphate. Currently, there is quite a range in the size of the T2S output of the T2S-expressing pathogens, going from one protein or activity as in K. pneumoniae, Pseudomonas alcaligenes, and V. parahaemolyticus to dozens as in Acinetobacter nosocomialis, B. pseudomallei, L. pneumophila, P. aeruginosa, and V. cholerae (Table 1). The output of many, if not all, T2S systems, however, will likely prove to be greater once proteomic analysis is applied. Most studies have identified T2S-dependent proteins in culture supernatants; however, there is increasing evidence that some substrates remain bound to the bacterial surface after secretion. The first such example was the pullulanase of K. oxytoca, and further examples have now been found in E. coli, P. aeruginosa, and V. vulnificus (Table 1). The mechanism by which the T2S apparatus facilitates the anchoring of proteins to the bacterial outer surface rely on acylation and hydrophobic or polar interactions (29). Nonetheless, by virtue of their surface localization, these proteins can be present on outer membrane vesicles (OMVs) that bleb from the bacterial cell surface (30). Other T2S substrates come to reside within OMVs, as a result of their localization in the periplasm prior to transport across the OM by the T2S apparatus (30). Because of their fusogenic capability, OMVs provide an alternative means for delivering T2S-associated substrates to host targets.

Collectively, the human pathogens that express T2S are responsible for a wide variety of diseases, ranging from pneumonia (A. baumannii, L. pneumophila, K. pneumoniae, P. aeruginosa, and S. maltophilia) to gastroenteritis and diarrhea (E. coli, V. cholerae, and Y. enterocolitica) to bloodstream (A. hydrophila, B. pseudomallei, and V. vulnificus), urinary tract (E. coli), and genital tract (C. trachomatis) infections (Table 1). Furthermore, these bacteria include both extracellular (Acinetobacter, Aeromonas, Escherichia, Klebsiella, Pseudomonas, Stenotrophomonas, and Vibrio species) and intracellular (Burkholderia species, C. trachomatis, L. pneumophila, and Y. enterocolitica) pathogens. These facts imply that T2S facilitates disease in a variety of ways and is not limited to a particular pathogenic event or site of infection. Support for this inference derives from the many types of degradative enzymes and toxins that are secreted by T2S; i.e., ADP-ribosylating enzymes, carbohydrate-degrading enzymes, lipolytic enzymes, nucleases, pore-forming proteins, phosphatases, peptidases, and proteases (Table 1). Particularly well-known examples of T2S-dependent substrates are cholera toxin produced by V. cholerae, exotoxin A of P. aeruginosa, and heat-labile (LT) toxin from enterotoxigenic E. coli. The most direct proof for the role of T2S in pathogenesis is based upon the attenuated virulence of T2S mutants in animal models of disease, as has been shown for A. baumannii, A. hydrophila, B. cenocepacia, B. pseudomallei, E. coli, K. pneumoniae, L. pneumophila, P. aeruginosa, V. vulnificus, and Y. enterocolitica (Table 1). Additional assays using these mutants and/or isolated secreted proteins have revealed a diversity of mechanisms by which T2S facilitates disease. These mechanisms include the death of host cells by lysis or toxicity, degradation of tissue and extracellular matrix, cleavage of defense molecules such as cytokines and complement components and other means of suppressing innate immunity, adherence to epithelial cell surfaces, disruption of the tight junctions between host cells, biofilm formation, invasion into host cells or subsequent intracellular growth, deubiquitination, iron acquisition, and other forms of nutrient assimilation, and alterations in host ion flux triggering diarrhea (Table 1). Undoubtedly, there are even more ways in which T2S promotes pathogenesis; e.g., proteomic analysis has revealed a number of T2S substrates that are “novel,” having no sequence similarity to known proteins or enzymes (e.g., L. pneumophila, P. aeruginosa, and V. cholerae) (Table 1).

T2S in pathogens of plants.

T2S systems are also present and functional in plant pathogens that belong to the gammaproteobacteria (Dickeya, Erwinia, Pectobacterium, Xanthomonas, and Xylella) and betaproteobacteria (Burkholderia and Ralstonia) (Fig. 1). The T2S-expressing phytopathogens include Burkholderia gladioli, Burkholderia glumae, Dickeya dadantii, Erwinia amylovora, Pectobacterium carotovorum, Pectobacterium wasabiae, Ralstonia solanacearum, Xanthomonas axonopodis, Xanthomonas campestris, Xanthomonas oryzae, and Xylella fastidiosa (Table 1). Collectively, they cause serious diseases of flowers, fruit (e.g., pear, citrus, and grape), rice, tubers, and vegetables (e.g., crucifers and peppers) (Table 1). Many of the concepts noted above when discussing the T2S-expressing human pathogens also apply here. For example, some of the plant pathogens have multiple T2S systems (D. dadantii, X. campestris pv. vesicatoria), secrete ≥15 T2S substrates (B. glumae, D. dadantii, P. carotovorum, and R. solanacearum), and express T2S substrates on their surface (D. dadantii). They also secrete some enzymes that are similar to those made by the human and animal pathogens (e.g., lipases and proteases) as well as “novel” proteins that may encode a new enzymatic activity and/or mediate a new type of process. Not surprisingly, the T2S systems of the phytopathogens elaborate a large number and variety of carbohydrate-degrading enzymes that specifically degrade plant tissue, e.g., cellulases, pectate lyases, xylanases, and polygalacturonases (Table 1). In every case examined, mutations in the genes encoding T2S diminish virulence in a relevant host(s) (Table 1), clearly showing the importance of T2S in plant pathogenesis.

T2S in nonpathogenic, environmental bacteria.

Although T2S in pathogens has received the greatest attention, there have been a number of studies documenting T2S functionality in nonpathogenic bacteria or bacteria that only very rarely cause disease (Table 1). These bacteria are quite diverse, ranging from alphaproteobacteria (Caulobacter and Gluconacetobacter) to betaproteobacteria (Cupriavidus and Ralstonia) to gammaproteobacteria (Aeromonas, Cellvibrio, Escherichia, Marinobacter, Methylococcus, Pseudoalteromonas, Pseudomonas, Shewanella, and Vibrio) to deltaproteobacteria (Geobacter) (Fig. 1). In most cases, they are primarily free-living organisms, inhabiting soil, freshwater, and/or salt water. However, some exist in symbiotic relationships with plants (Gluconacetobacter diazotrophicus and Pseudomonas fluorescens) or animals (Aeromonas veronii, E. coli, and Vibrio fischeri), and in the case of A. veronii, T2S actually promotes the symbiosis with leeches (Table 1). Based on the genome database, it is likely that many more nonpathogens utilize T2S, including species of Azoarcus, Bdellovibrio, Bradyrhizobium, Chromobacterium, Mesorhizobium, Methylotenera, and Myxococcus as well as marine gammaproteobacteria belonging to Idiomarina, Marinomonas, Psychromonas, and Saccharophagus (10, 18, 31–33) (Fig. 1). The study of nonpathogens has revealed a variety of secreted proteins and processes that had not been seen with the pathogenic organisms. Among the novel T2S-dependent substrates are the multicopper oxidase of Geobacter sulfurreducens, levansucrase of G. diazotrophicus, c-type cytochrome of Methylococcus capsulatus, Mn-oxidizing enzymes of Pseudomonas putida, and NAD-glycohydrolases of V. fischeri, and included in the T2S-facilitated processes are pigmentation by Pseudomonas tunicata and Fe³⁺ reduction and extracellular respiration by Shewanella oneidensis (Table 1). Thus, by considering the full range of T2S-expressing bacteria, the functional diversity of T2S can be even better appreciated.

T2S in the transition of environmental bacteria to pathogens, as illustrated by V. cholerae and L. pneumophila.

Nearly all of the T2S-expressing pathogens exist in the environment in addition to their higher organism hosts. Arguably, the impact of T2S is best appreciated by contemplating how T2S assists bacteria in both their environmental niche(s) and their human, animal, or plant host(s). This point is most clear from studies done with V. cholerae, the agent of cholera and a classic extracellular pathogen, and L. pneumophila, the etiologic agent of Legionnaires' disease and a well-known intracellular pathogen. In the case of V. cholerae, the Eps T2S system enhances attachment to and biofilm formation on abiotic and biotic surfaces in marine environments (34). This, in turn, promotes the growth of planktonic V. cholerae as well as bacterial colonization of marine creatures such as bivalves, copepods, and cladocerans (35). Among the T2S-dependent proteins that mediate environmental persistence are the chitin-binding protein GbpA that aids in attachment, ChiA and other chitinases that generate carbon and energy sources for growth, the biofilm-promoting RbmC, and the HapA protease which can degrade the matrix that covers the eggs of chironomids (34–37). By helping to increase the numbers of V. cholerae in the environment, T2S promotes the transmission of the Vibrio pathogen to human hosts via the ingestion of contaminated waters. Once in the human host, T2S continues to play a major role by secreting HapA which degrades mucin and thereby permits bacterial access to the underlying intestinal epithelium, GbpA which enhances binding to mucins that overlay the epithelium, cholera toxin which triggers water efflux from enterocytes (i.e., massive watery diarrhea), and HapA, VesA, and VesB which can proteolytically activate cholera toxin and other toxins (34, 35, 38–41). In summary, T2S is unquestionably important for V. cholerae both in its natural marine environment and in the human host, facilitating, in multiple ways, extracellular replication and dissemination (Fig. 2A).

FIG 2.

Roles of T2S in V. cholerae and L. pneumophila. (A) More than 20 proteins are secreted via the T2S system of V. cholerae. T2S promotes the environmental survival of extracellular V. cholerae in a variety of ways, including the colonization of biotic surfaces (left side, in blue). This facilitates transmission to the human host, where T2S mediates another set of activities that leads to cholera (right side, in red). (B) More than 25 substrates are handled by the T2S system of L. pneumophila. In the environment, T2S facilitates the spread of L. pneumophila by contributing to planktonic survival, biofilm formation, and intracellular infection of amoebae (left side, in green). Following the inhalation of L. pneumophila, T2S promotes bacterial growth within lung macrophages, which leads to tissue damage and pneumonia (right side, in red).

Turning to L. pneumophila, it is necessary to first emphasize that the persistence of the Legionella pathogen in freshwater environments is primarily due to its capacity to infect a wide array of amoebae (42). The Lsp T2S system of L. pneumophila has a major role in infection of amoebae, promoting intracellular growth in at least four genera, i.e., Acanthamoeba, Naegleria, Vermamoeba (formerly Hartmannella), and Willaertia (43–47). This function of T2S is manifest over a temperature range of 22 to 37°C, further indicating the impact of T2S across different aquatic niches (48). The T2S-dependent substrates that are known to potentiate amoebal infection are the acyltransferase PlaC, metalloprotease ProA, RNase SrnA, and novel proteins NttA and NttC (46, 47, 49, 50). Interestingly, the importance of each of these secreted proteins varies depending upon the amoeba being infected, suggesting that the T2S repertoire of L. pneumophila has evolved, in part, to enhance the bacterium's broad host range (47). Besides its predilection for amoebae, L. pneumophila survives extracellularly in its aquatic habitats, either planktonically or in multiorganismal biofilms (51, 52). T2S is also relevant for these lifestyles, as documented in several ways. First, T2S mutants display impaired extracellular survival in tap water samples when incubated at 4 to 25°C (48). The fact that the secretome of L. pneumophila changes with temperature suggests that one or more secreted proteins, including a predicted peptidyl-prolyl cis-trans isomerase (PPIase), facilitate low-temperature survival (53). Second, a mutant specifically lacking the T2S-dependent Lcl protein exhibits a reduced ability to form biofilms (54). Finally, T2S mutants demonstrate impaired sliding motility, which is linked to the secretion of a novel surfactant (55–57). By fostering L. pneumophila growth within water systems, T2S contributes to the genesis of human infection which occurs via the inhalation of contaminated water droplets generated by various aerosol-generating devices. Yet, T2S also enhances L. pneumophila growth within the lung itself; i.e., secretion mutants are impaired in both murine and guinea pig models of pneumonia (26, 43, 58). The intrapulmonary role of T2S primarily involves L. pneumophila intracellular infection of macrophages (26, 59). Recent studies indicate that T2S is not required for L. pneumophila entry into the macrophage host or its subsequent evasion of phagosome-lysosome fusion (60). Rather, T2S facilitates the onset of bacterial replication at 4 to 8 h postentry as well as the capacity to grow to large numbers within the Legionella-containing vacuole at 12 h and beyond. This growth promotion involves both the retention of the host GTPase Rab1B on the Legionella-containing vacuole as well as a Rab1B-independent event(s) that is yet to be defined (60). Besides facilitating bacterial growth in macrophages, T2S is necessary for optimal replication within epithelial cells, which likely are a secondary host cell during lung infection (59). Furthermore, the T2S system dampens the cytokine output of infected macrophages and epithelial cells (59). This suppression of the innate immune response, which is manifest at the transcriptional level due to dampening of the MyD88 and Toll-like receptor 2 signaling pathway, is believed to initially limit inflammatory cell infiltrates into the lung and thus permit prolonged bacterial growth (61). As for the T2S-dependent proteins that are known to potentiate disease, the chitinase ChiA promotes bacterial growth and persistence in the lungs but in a manner that appears to be independent of intracellular growth (58). One hypothesis for this novel finding is that ChiA acts upon chitin-like molecules (e.g., O-GlcNAcylated proteins) in the lung. Finally, the metalloprotease ProA functions as a virulence factor by degrading lung tissue and cytokines (59, 62–64). Thus, L. pneumophila provides a striking example of the many ways in which T2S can promote both bacterial growth in the environment and virulence in the human host (Fig. 2B). L. pneumophila's adaptation to an intracellular niche in aquatic amoebae engendered it with the capacity to grow in human macrophages, and it is now clear that T2S plays a major role in both forms of intracellular infection.

Final thoughts and ongoing questions.

In recent years, we have experienced an impressive increase in knowledge about bacterial T2S. These advancements include not only the fine-structure analysis of the T2S apparatus but also, as detailed here, a refined understanding of the distribution of T2S among Gram-negative organisms and the large and diverse roles of this secretion system (Table 1). Given the breadth of its involvement in pathogenic processes, it is clear that the importance of T2S rivals that of the other known secretion systems operating in Gram-negative bacteria that afflict humans, animals, or plants. Although we have learned a great deal about the output and functional consequences of T2S in pathogens and nonpathogens, there is still much insight to be gained, given that many of the secreted factors produced by these bacteria are still only minimally defined or entirely uncharacterized (Table 1). Indeed, some of these T2S-dependent substrates may represent new types of enzymes which might mediate novel pathogenic activities. Based on the data assembled in Table 1, there are also a number of T2S systems that are only slightly characterized and/or not yet examined in pertinent disease models. Moreover, the genome database indicates that there are many other bacteria, including pathogens, that harbor T2S systems that have not been investigated at all. All of these studies should take into consideration how the output and function of a T2S system might change depending upon growth conditions and regulatory networks. As the T2S catalog expands, various comparisons between the secreted proteins might reveal new structural similarities or motifs that help address a long-standing question in the field, that is, how T2S substrates are recognized by the secretion apparatus. In light of the now-demonstrated importance of T2S in a wide range of pathogenic bacteria, future work should also consider using the structural and functional knowledge gained to develop potential new strategies or reagents for preventing or combatting human, animal, or plant infections.