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 theProteobacteria, occurring in many, but not all, genera in theAlphaproteobacteria,Betaproteobacteria,Gammaproteobacteria, andDeltaproteobacteriaclasses. Prominent human and/or animal pathogens that express a T2S system(s) includeAcinetobacter baumannii,Burkholderia pseudomallei,Chlamydia trachomatis,Escherichia coli,Klebsiella pneumoniae,Legionella pneumophila,Pseudomonas aeruginosa,Stenotrophomonas maltophilia,Vibrio cholerae, andYersinia enterocolitica. T2S-expressing plant pathogens includeDickeya dadantii,Erwinia amylovora,Pectobacterium carotovorum,Ralstonia solanacearum,Xanthomonas campestris,Xanthomonas oryzae, andXylella 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. pneumophilaemploys 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.

Type II protein secretion is critical for Legionella pneumophila infection of amoebae, macrophages, and mice. Previously, we found several enzymes to be secreted by this (Lsp) secretory pathway. To better define the L. pneumophila type II secretome, a 2D electrophoresis proteomic approach was used to compare proteins in wild-type and type II mutant supernatants. We identified 20 proteins that are type II-dependent, including aminopeptidases, an RNase, and chitinase, as well as proteins with no homology to known proteins. Because a chitinase had not been previously reported in Legionella , we determined that wild type secretes activity against both p -nitrophenyl triacetyl chitotriose and glycol chitin. An lsp mutant had a 70–75% reduction in activity, confirming the type II dependency of the secreted chitinase. Newly constructed chitinase ( chiA ) mutants also had ≈75% less activity, and reintroduction of chiA restored the mutants to normal levels of activity. Although chiA mutants were not impaired for in vitro intracellular infection, they were defective upon intratracheal inoculation into the lungs of A/J mice, and antibodies against ChiA were detectable in infected animals. In contrast, mutants lacking a secreted phosphatase, protease, or one of several lipolytic enzymes were not defective in vivo . In sum, this study shows that the output of type II secretion is greater in magnitude than previously appreciated and includes previously undescribed proteins. Our data also indicate that an enzyme with chitinase activity can promote infection of a mammalian host.

Legionella pneumophila , the gram-negative agent of Legionnaires' disease, possesses type IV pili and a type II protein secretion (Lsp) system, both of which are dependent upon the PilD prepilin peptidase. By analyzing multiple pilD mutants and various types of Lsp mutants as well as performing trans -complementation of these mutants, we have confirmed that PilD and type II secretion genes are required for L. pneumophila infection of both amoebae and human macrophages. Based upon a complete analysis of lspDE , lspF , and lspG mutants, we found that the type II system controls the secretion of protease, RNase, lipase, phospholipase A, phospholipase C, lysophospholipase A, and tartrate-sensitive and tartrate-resistant acid phosphatase activities and influences the appearance of colonies. Examination of the developing L. pneumophila genome database indicated that the organism has two other loci ( lspC and lspLM ) that are predicted to promote secretion and thus a set of genes that is comparable to the type II secretion genes in other gram-negative bacteria. In contrast to lsp mutants, L. pneumophila pilus mutants lacking either the PilQ secretin, the PspA pseudopilin, or pilin were not defective for colonial growth, secreted activities, or intracellular replication. L. pneumophila dot/icm mutants were also not impaired for type II-dependent exoenzymes. Upon intratracheal inoculation into A/J mice, lspDE , lspF , and pilD mutants, but not pilus mutants, exhibited a reduced ability to grow in the lung, as measured by competition assays. The lspF mutant was also defective in an in vivo kinetic assay. Examination of infected mouse sera revealed that type II secreted proteins are expressed in vivo. Thus, the L. pneumophila Lsp system is a virulence factor and the only type II secretion system linked to intracellular infection.

In order to determine the role of ferrous iron transport inLegionellapathogenesis, we identified and mutated thefeoBgene in virulentLegionella pneumophilastrain 130b. As it is inEscherichia coli, theL. pneumophila feoBgene was contained within a putativefeoABoperon.L. pneumophila feoBinsertion mutants exhibited decreased ferrous but not ferric iron uptake compared to the wild type. Growth on standard buffered charcoal yeast extract agar or buffered yeast extract broth was unaffected by the loss ofL. pneumophilaFeoB. However, theL. pneumophila feoBmutant had a reduced ability to grow on buffered charcoal yeast extract agar with a reduced amount of its usual iron supplementation, a phenotype that could be complemented by the addition offeoBintrans. In unsupplemented buffered yeast extract broth, thefeoBmutant also had a growth defect, which was further exacerbated by the addition of the ferrous iron chelator, 2,2′-dipyridyl. ThefeoBmutant was also 2.5 logs more resistant to streptonigrin than wild-type 130b, confirming its decreased ability to acquire iron during extracellular growth. Decreased replication of thefeoBmutant was noted within iron-depletedHartmannella vermiformisamoebae and human U937 cell macrophages. The reduced intracellular infectivity of thefeoBmutant was complemented by the introduction of a plasmid containingfeoAB. TheL. pneumophila feoBgene conferred a modest growth advantage for the wild type over the mutant in a competition assay within the lungs of A/J mice. Taken together, these results indicate thatL. pneumophilaFeoB is a ferrous iron transporter that is important for extracellular and intracellular growth, especially in iron-limited environments. These data represent the first evidence for the importance of ferrous iron transport for intracellular replication by a human pathogen.

The virulence of Legionella pneumophila is dependent upon its capacity to acquire iron. To identify genes involved in expression of its siderophore, we screened a mutagenized population of L. pneumophila for strains that were no longer able to rescue the growth of a ferrous transport mutant. However, an unusual mutant was obtained that displayed a strong inhibitory effect on the feoB mutant. Due to an insertion in hmgA that encodes homogentisate 1,2-dioxygenase, the mutant secreted increased levels of pyomelanin, the L. pneumophila pigment that is derived from secreted homogentisic acid (HGA). Thus, we hypothesized that L. pneumophila -secreted HGA-melanin has intrinsic ferric reductase activity, converting Fe ³⁺ to Fe ²⁺ , but that hyperpigmentation results in excessive reduction of iron that can, in the case of the feoB mutant, be inhibitory to growth. In support of this hypothesis, we demonstrated, for the first time, that wild-type L. pneumophila secretes ferric reductase activity. Moreover, whereas the hyperpigmented mutant had increased secreted activity, an lly mutant specifically impaired for pigment production lacked the activity. Compatible with the nature of HGA-melanins, the secreted ferric reductase activity was positively influenced by the amount of tyrosine in the growth medium, resistant to protease, acid precipitable, and heterogeneous in size. Together, these data represent the first demonstration of pyomelanin-mediated ferric reduction by a pathogenic bacterium.