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Invited review: Breaking barriers attack on innate immune defences by omptin surface proteases of enterobacterial pathogens Johanna Haiko, Marjo Suomalainen, Teija Ojala, Kaarina Lähteenmäki and Timo K. Korhonen Innate Immunity 2009; 15; 67 DOI: 10.1177/1753425909102559 The online version of this article can be found at: http://ini.sagepub.com/cgi/content/abstract/15/2/67

Published by: http://www.sagepublications.com

On behalf of: International Endotoxin & Innate Immunity Society

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Invited review

Breaking barriers – attack on innate immune defences by omptin surface proteases of enterobacterial pathogens

15(2) (2009) 67–80 ß SAGE Publications 2009 ISSN 1753-4259 (print) 10.1177/1753425909102559

Johanna Haiko1, Marjo Suomalainen1, Teija Ojala1, Kaarina La¨hteenma¨ki2, Timo K. Korhonen1 1 2

General Microbiology, Faculty of Biosciences, University of Helsinki, Helsinki, Finland Research and Development, Red Cross Finland Blood Service, Helsinki, Finland

The omptin family of Gram-negative bacterial transmembrane aspartic proteases comprises surface proteins with a highly conserved b-barrel fold but differing biological functions. The omptins OmpT of Escherichia coli, PgtE of Salmonella enterica, and Pla of Yersinia pestis differ in their substrate specificity as well as in control of their expression. Their functional differences are in accordance with the differing pathogenesis of the infections caused by E. coli, Salmonella, and Y. pestis, which suggests that the omptins have adapted to the life-styles of their host species. The omptins Pla and PgtE attack on innate immunity by affecting the plasminogen/plasmin, complement, coagulation, fibrinolysis, and matrix metalloproteinase systems, by inactivating antimicrobial peptides, and by enhancing bacterial adhesiveness and invasiveness. Although the mechanistic details of the functions of Pla and PgtE differ, the outcome is the same: enhanced spread and multiplication of Y. pestis and S. enterica in the host. The omptin OmpT is basically a housekeeping protease but it also degrades cationic antimicrobial peptides and may enhance colonization of E. coli at uroepithelia. The catalytic residues in the omptin molecules are spatially conserved, and the differing polypeptide substrate specificities are dictated by minor sequence variations at regions surrounding the catalytic cleft. For enzymatic activity, omptins require association with lipopolysaccharide on the outer membrane. Modification of lipopolysaccharide by in vivo conditions or by bacterial gene loss has an impact on omptin function. Creation of bacterial surface proteolysis is thus a coordinated function involving several surface structures. Keywords: surface proteolysis, plague, salmonellosis, Escherichia coli, plasminogen activator, gelatinase, antimicrobial peptides

INTRODUCTION Surface-located or soluble extracellular proteases have an important role in bacterial resistance against innate and acquired immune defences of infected mammals, and they are virulence factors in several invasive bacterial infections. Bacterial proteases affect immunological defence functions at various stages of the infectious processes, e.g. through degradation of circulating antibodies or complement proteins as well as antimicrobial peptides in epithelial or phagocytic cells.

Bacterial proteases may also directly damage host structures, such as fibrin clots or extracellular matrices that prevent bacterial migration within the host. Several pathogenic bacterial species have the capacity to engage host proteolytic or homeostatic systems and to turn them to advance bacterial spread across tissue barriers.1–4 Bacterial proteases can be multifunctional and also exhibit functions that are not proteolytic in nature, such as adhesiveness or invasiveness to subepithelial tissue or epithelial and phagocytic cells. On the other hand, engagement of host-derived proteolytic activity

Received 24 October 2008; Revised 29 December 2008; Accepted 3 January 2009 Correspondence to: Prof. Timo K. Korhonen, General Microbiology, Faculty of Biosciences, University of Helsinki, FI-00014 Helsinki, Finland. Tel: þ358 9 19159260; E-mail: [email protected] Downloaded from http://ini.sagepub.com at MHH-Bibliothek on April 27, 2009

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Haiko, Suomalainen, Ojala, La¨hteenma¨ki, Korhonen S. flexneri SopA E. coli OmpT E. coli OmpP S. enterica PgtE Y. pestis YcoA Y. pseudotuberculosis YcoB

E. pyrifoliae Epo Enterobacter sp. Y. pestis Pla

L. pneumophila Leo

V. fischeri

D. psycrophila 0.1

M. loti

Fig. 1. Evolutionary tree of omptins. The sequences of omptins from Shigella flexneri, Escherichia coli, Yersinia pestis, Yersinia pseudotuberculosis, Legionella pneumophila, Vibrio fischeri, Mesorhizobium loti, Desulfotalea psycrophila, Enterobacter spp., Erwinia pyrifoliae, and Salmonella enterica were from the omptin family A26 of the MEROPS database for proteolytic enzymes.6 The phylogenetic and molecular evolutionary analyses were conducted using MEGA version 4.120

may involve seemingly ‘bystander’ surface structures, such as fimbriae, flagella, or surface proteins traditionally considered cytoplasmic metabolic enzymes, such as enolase.2,5 This review addresses functions and mechanisms in how a conserved surface-located protease fold, the omptin family of aspartic proteases, has diverged in pathogenic enteric bacteria to support their differing lifestyles and avoidance of host innate immune defences. Omptin proteases have been detected in at least 12 Gram-negative bacterial species that infect either mammalians or plants6 (Fig. 1). Here, we will concentrate on the three best known of them: OmpT of Escherichia coli, Pla of Yersinia pestis, and PgtE of Salmonella enterica. The plague bacterium Y. pestis causes a highly fatal, flea-borne zoonosis, plague, where the bacterium spreads from the subdermal injection site into lymphatic tissue. Swelling of lymph nodes causes buboes that are characteristic for the disease, and the bacterium may subsequently invade the circulation as well as the lungs and other organs.7 The pla gene is associated with highly invasive strains and pandemic lineages of Y. pestis.8–10 Early studies identified Pla as a major virulence factor in bubonic plague, where the bacteria invade from the intradermal flea-bite site to lymphatic vessels and to regional draining lymph nodes and then multiply to cause development of buboes.7 Deletion of pla causes a million-fold increase in LD50 of Y. pestis in subcutaneously infected mice, whereas no difference is seen in intravenously infected mice.11 A recent study showed that Pla is specifically needed for establishment of bubonic plague in animals infected via intradermal bites by infected fleas, whereas Pla is not essential in primary septicemic plague where the bacteria are

injected directly into capillaries.10 These findings indicate that metastasis of Y. pestis from intradermal tissue to lymph nodes specifically depends on Pla. More recently, the essential role of Pla in pneumonic plague was also demonstrated.12 In pneumonic plague, the bacteria are transmitted to lungs within aerosols from humans to humans, and Pla allows Y. pestis to multiply in the airways, causing lethal fulminant pneumonia. Thus, the phenotypes attributed to Pla are surprisingly distinct in the two disease models of plague: in bubonic plague, Pla enhances bacterial migration to lymph nodes, whereas in pneumonic plague, Pla enables localized growth. The gastroenteritis-associated pathogen S. enterica is an oral-faecal pathogen that invades the epithelium in the ileum, and the subsequent systemic spread of the bacterium is mainly intracellular within infected phagocytes.13 The omptin PgtE of S. enterica has so far been characterized only in the serovar Typhimurium but a conserved pgtE gene is also present in all S. enterica serovar Typhi, Paratyphi and Choleraesuis strains sequenced so far.6 Protease PgtE enhances virulence of Typhimurium: deletion of pgtE impairs bacterial survival in mice, in murine macrophages, and in human serum.14–16 After intraperitoneal infection in mice, deletion of pgtE causes an approximately 10-fold reduction in the numbers of bacteria in the spleen and the liver.16 Thus, the effect of PgtE on survival of Salmonella in the host is significant, but considerably smaller than that described for Pla in bubonic plague. This probably reflects the fact that Salmonella possesses a number of virulence factors with partially overlapping functions. The numerous E. coli pathogroups differ in virulence gene content as well as in the degree of bacterial invasiveness within the host, and are associated with various forms of intestinal infections, urinary tract infection, as well as systemic sepsis potentially leading to newborn meningitis.17 The ompT gene shows a slightly elevated prevalence in E. coli strains associated with extra-intestinal infections; however, there is no clear association of OmpT with clinical strains or a pathogenic group of E. coli.18 A pathogenetic role for OmpT remains open; it appears to be a housekeeping protease functioning in turnover and degradation of endogenous or fusion proteins that come into contact with the outer membrane. The omptins in these three bacterial species offer a prime example of evolution and adaptation of a protein fold to support differing bacterial life-styles and pathogenetic mechanisms. This adaptation involves minor sequence variation at critical protein sites that leads to differences in polypeptide specificity of proteolysis, as well as differential control of omptin gene expression. A further issue in this review is that the successful

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Attack on innate immune defences by omptin surface proteases of enterobacterial pathogens 69 proteolytic attack on innate immunity by these bacteria involves, in addition to the omptin proteases themselves, other cell surface structures. In particular, modification of the lipopolysaccharide (LPS) structure is critical both for omptin function and for bacterial avoidance of host innate immune defences. For a general overview of omptin structure and function, the reader is referred to two recent reviews.18,19

L3 L4 Asp86

L5 Lys262

L2 Asp84 Asp206 L1

OMPTIN PROTEASES: A HORIZONTALLY SPREAD SURFACE PROTEASE FOLD IN GRAM-NEGATIVE BACTERIA The predicted over-all amino acid sequences encoded by the omptin genes are 50–70% identical, and the structure predictions indicate a conserved transmembrane protein fold, which has apparently spread by horizontal gene transfer (HGT) amongst Gram-negative bacterial species.16,18–21 Many of the omptin genes recognized so far are encoded by plasmids or other transferable elements, and also other features, such as the mosaic nature of the plasmids, indicate a role for HGT in their spread. The pla gene is located on the Y. pestis-specific 9.5-kb plasmid pPCP1, pgtE on the chromosome, and ompT on a prophage.22–24 The mature form of the predicted Pla and PgtE proteins share 75% amino acid sequence identity;16,21,25 sequence analyses indicate that Pla and PgtE belong to the same omptin subfamily (Fig. 1),26 suggesting that they share a common recent ancestor. The pla gene is specific to Y. pestis and lacking in the human pathogenic species Yersinia enterocolitica and Yersinia pseudotuberculosis.22 The plague bacterium Y. pestis has diverged from the intestinal pathogen Y. pseudotuberculosis only some 5000 years ago,27,28 which indicates that the acquisition of pla or its ancestor form by Y. pestis is a recent event in the evolution of this species. On the other hand, the predicted sequence of OmpT is less related to those of Pla and PgtE, and OmpT belongs to a different omptin subfamily (Fig. 1),26 which together with the different genomic localization indicates that the divergence of OmpT and Pla/PgtE is an early event, perhaps associated with the separation of E. coli and Salmonella as species some 120 million years ago.29 Thus, the highly virulent Pla seems to be a recent member in the omptin family, which has drawn interest in its evolution into a dramatic virulence factor.20,30 The crystal structure of OmpT has been resolved and provides a structure platform for the other omptins.16,20,21,31 The omptin molecules transverse the ˚ long b-barrel outer membrane and share a common 70-A fold with 10 antiparallel transmembrane b-strands, five surface-exposed loops (L1–L5) and five periplasmic turns (Fig. 2). The omptin barrel protrudes

His208

Arg138 Arg171 Glu136 Tyr134

Fig. 2. The b-barrel structure of Pla of Y. pestis. The catalytic residues Asp84, Asp86, Asp206, and His208 are shown in green, the putative LPSbinding residues Tyr134, Glu136, Arg138, and Arg171 in red, and the autoprocessing site Lys262 in blue. L1–L5 denote the mobile surface loops of Pla. The structure was modelled using the OmpT crystal structure (pdb id: 1i78)31 as a template with the MODELLER program.121 The visualization was done with the VMD program.122

˚ from the outer membrane extracellularly about 40 A lipid bilayer.31 Mature omptins have 290–300 amino acids and their catalytic residues are spatially completely conserved: Asp84, Asp86, Asp206, and His208 (Fig. 2). As expected for a transmembrane protein, the insertions and deletions that give the slight variation in molecular size, are located in the flexible outer loop L1–L5 structures of the omptin molecules. The omptins also autoprocess themselves; in Pla, the autoprocessing occurs at residue K262 in L5 (Fig. 2).20 This residue is not conserved in OmpT and PgtE, and OmpT is autoprocessed at another non-conserved lysine residue, K217 in L4 closer to the catalytic site.32 In the PgtE molecule, the probable autoprocessing site is at residue R154.21 The autoprocessing seems not to take place in every omptin molecule in the cell wall,20 and its biological role remains open. Earlier, omptins were classified as serine proteases; however, on the basis of OmpT crystal structure, they were reclassified as aspartic proteases.6,31 The proteolytic mechanism of omptins combines features of serine and aspartic proteases and involves conserved His208–Asp206 dyad and Asp84–Asp86 couple as catalytic residues (Fig. 2). The His–Asp dyad activates a water molecule and nucleophilically attacks the substrate at the scissile bond, thus functionally resembling the Ser–Asp– His catalytic triad of serine proteases. The Asp–Asp couple is characteristic of aspartic proteases and participates either in proton translocation or stabilization of an oxyanion intermediate. Protease OmpT prefers substrate cleavage next to a basic amino acid: arginine or lysine is preferred at the P1 site. Requirements for

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Haiko, Suomalainen, Ojala, La¨hteenma¨ki, Korhonen

the P10 and the other P/P0 residues are more flexible, but OmpT prefers positively charged amino acids in the vicinity of the cleavage site, and Pla has a preference towards Arg–Val bond in the plasminogen molecule. Affinity for the substrates is also affected by ionic interactions; the presence of negatively charged amino acids around the active site of OmpT probably increases its activity towards basic target residues.31

LIPOPOLYSACCHARIDE IS CRITICAL FOR THE ACTIVITY OF OMPTINS Recent studies have shown that Y. pestis and S. enterica modify their LPS structure under host conditions.33 These modifications have a profound effect on bacterial resistance against innate immune responses as well as on omptin functions. Endotoxic LPS is an immunodominant surface molecule and a major bacterial virulence factor in infections by Gram-negative bacteria, including those caused by E. coli, S. enterica as well as Yersinia spp. The structure of the LPS consists of three regions: the O-antigen and the core which both consist of (poly)saccharide chains, and lipid A composed of a glucosamine disaccharide substituted with fatty acids and phosphate. The long O-antigen chains of smooth LPS are important virulence determinants that contribute to enterobacterial colonization in the gut and to their resistance to killing by neutrophils as well as by the complement system of normal serum.34 O-Antigen is also highly immunogenic and associated with structural heterogeneity as well as inter- and intrastrain variability34 giving rise to the antigenic variation that helps to avoid acquired immunity defences. On the other hand, lipid A, which is responsible for the endotoxin activity of LPS, is a conserved structure and a pathogen-associated molecular pattern recognized by Toll-like receptor 4 (TLR4) in mammalian cells. The binding of LPS to TLR4 leads to activation of the transcription factor nuclear factor-kB (NF-kB) and to increased transcription of pro-inflammatory cytokines.35 This elicits a highly potent inflammatory response, which is critical for elimination of the pathogen, but can sometimes be harmful, and even lethal, for the host. A consensus motif for lipid A binding has been identified in three-dimensional structure of LPS-binding proteins from prokaryotes as well as eukaryotes.36 In the human MD-2 protein, which presents LPS to TLR4, the interaction is dependent on two spatially nearby arginines that bind to 40 -phosphate in lipid A.37 The importance of the two arginines was first detected in the crystal structure of E. coli FhuA outer membrane protein complexed with LPS.38 A similar spatial arrangement of two arginines, R138 and R171 in

b-strands 5 and 6, is evident also in the structure of omptins (Fig. 2). Indeed, purified His-tagged OmpT, Pla and PgtE require exogenous LPS to be active,20,21,32,39 and substitution of the two arginines to glutamates leads to significant decrease in the proteolytic activity of PgtE overexpressed in S. enterica.21 These findings indicate that the interaction with LPS is functionally critical for omptins. Interestingly, this may explain their occurrence in Gram-negative bacteria only. In addition to arginines 138 and 171, the omptin–LPS interaction probably also involves the conserved residues Tyr134 and Glu136 in b-strand 5, whose counterparts in the FhuA protein were identified by crystallography to be in contact with lipid A fatty acids and phosphates.31,36,39 Exactly how LPS binding enhances omptin activity has not been defined. Kramer and co-workers39 did not detect by circular dichroism spectroscopy any gross conformational changes in OmpT upon LPS-binding, and it seems that the binding of LPS in the outer membrane has a minor conformational effect that orients the omptin surface loops, in particular the large L3 between b-strands 5 and 6 (Fig. 2), in a stable conformation which enhances correct recognition of polypeptide substrates. The omptins Pla, OmpT and PgtE require rough LPS for activity but are inhibited by the long O-antigen chains in smooth LPS.14,20,21 The inhibition by O-chains is not surprising in view of the intimate contact of omptins with LPS, and it appears that it results from a steric inhibition of substrate recognition by the long and mobile O-chains. The two severe pathogens in this review deal differently with this problem: Y. pestis does not produce smooth LPS because the O-antigen biosynthesis genes are inactivated, whereas S. enterica modifies its LPS and PgtE expression inside macrophages (see discussion below).14,40 Evolution of Y. pestis from the gastrointestinal pathogen Y. pseudotuberculosis serogroup O1b is associated with several genetic changes, including point mutations as well as loss and gain of genes; one of these genetic events is the inactivation of the genes encoding the O-antigen synthesis.40 Thus Y. pestis possesses a short-chain, rough type of LPS. On the other hand, the O-antigen is a determinant of virulence in Y. pseudotuberclosis, Y. enterocolitica, and S. enterica, and its loss subjects these bacteria to complementmediated and phagocytic killing,41–45 which has led to the question of the selective advantage of the loss of the O-antigen. Rough strains of Y. pestis are resistant to complement-mediated killing46 and the high proteolytic activity of Pla in the absence of steric inhibition by O-chains is clearly one of the pathogenetic advantages of rough LPS in Y. pestis.21 In S. enterica serovar Typhimurium, the number of O-antigen subunits in smooth LPS preparations

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Attack on innate immune defences by omptin surface proteases of enterobacterial pathogens 71 varies from zero to 480, and the distribution of O-chain lengths is clustered around a modal length controlled by the wzz genes.47–50 Deletion of the wzz genes leads to short-length O-chains, attenuates Typhimurium in the mouse and makes the bacterium susceptible to complement.50 Reduced transcription of the rfb genes, which encode enzymes functioning in O-antigen assembly, were observed in Typhimurium cells from mouse macrophages,51 and modification of LPS to a shorter O-chain length was indeed observed in Typhimurium cells isolated from Salmonella containing vesicles of mouse macrophages.14 These bacteria also expressed high PgtE proteolytic activity not seen in bacteria cultivated in laboratory culture media. It appears that the control of the O-chain length involves the PhoP/Q sensory system,52,53 which also controls structural changes in lipid A and, as discussed below, expression of PgtE. The PhoP/Q-regulated structural changes include addition of palmitate, aminoarabinose, and phosphoethanolamine to lipid A, as well as hydroxylation and de-acylation of specific fatty acids.35 These lipid A modifications enhance intracellular survival of Salmonella in mouse macrophages by promoting resistance to antimicrobial peptides. In addition, the PhoP/ Q-regulated lipid A structures show reduced stimulation in mouse and human cell lines expressing the human TLR4 complex.52,54 In Y. pestis cells, growth temperature and environmental pH affect the degree of acylation in lipid A and the level of substitution of phosphate groups with aminoarabinose. Upon a shift from 25 C (i.e. the growth temperature in the fleas) to the mammalian temperature 37 C, the lipid A mixture of hexa-, penta-, and tetra-acylated forms is modified into a predominantly tetra-acylated lipid A.55–58 Expression of the lpxM and lpxP genes which encode acyltransferases that add C12 and C16:1 groups to the lipid A precursor and produce the hexa-acylated lipid A, is up-regulated at 21 C.59 Further temperature-induced changes in LPS of Y. pestis include lower content of aminoarabinose in lipid A from 37 C as well as differences in terminal core monosaccharides.56,60,61 The acylation level in lipid A greatly affects its capacity to activate the innate immune cell responses;62 indeed, Y. pestis LPS from bacteria grown at 27 C is a stronger inducer of tumor necrosis factor a (TNF-a) in mouse and human macrophages than the LPS from bacteria grown at 37 C.56 This LPS modification enables Y. pestis to evade detection by TLR4 in human cells and the subsequent induction of pro-inflammatory cytokines. Montminy and co-workers63 genetically modified Y. pestis to produce hexa-acylated LPS only; the resulting modified strain induced cellular responses and was completely avirulent in subcutaneously infected mice.63 These results show that the evasion of the LPSinduced TLR4 activation and inflammatory response

through lipid A alteration is an essential factor in the virulence of the plague bacterium. The acylation level in lipid A may also affect the function of Pla. The low acylation in LPS is associated with increased fluidity of acyl chains as well as with higher permeability to hydrophobic probes.64 This indicates that outer membrane proteins, such as Pla, are more mobile in the outer membrane lipid bilayer at higher temperature, which can be involved in the observed transcription-independent higher activity of Pla in cells from 37 C.

EXPRESSION OF OMPTINS Transcription of pla in Y. pestis is controlled by the cAMP receptor protein, which controls non-glucose metabolism in enterobacterial species.65 Consequently, expression of Pla is partially repressed in glucosecontaining growth medium. The plague bacterium ferments and utilizes a number of different catabolites for growth;66 it is interesting that regulation of Pla expression is linked with expression of metabolic activities essential for host colonization.65 The expression of Pla is high in laboratory media, and pla, although not transcriptionally induced, is one of the most highly expressed genes in Y. pestis cells from buboes of infected mice.10 Transfer of Y. pestis from the flea to the human host involves a change in growth temperature, from 25 C to 37 C, which has led to assessment of a possible temperature effect on Pla expression and activity. A marginal increase in the amount of Pla in cells from 37 C was detected in a proteomic study.67 Transcriptome analysis of Y. pestis showed no induction of pla at 37 C in human plasma.68 However, proteolytic activity by Y. pestis is higher in cells from 37 C,69,70 and it seems probable that this results from cell wall changes, such as structural changes in LPS, that indirectly involve Pla. In contrast to Pla in Y. pestis, PgtE is not expressed by wild-type Salmonella cultivated in laboratory culture media.14,21 Expression of PgtE is regulated by the SlyA transcriptional regulator, which in turn is regulated by the PhoP/Q regulatory system.71 The genes activated by PhoP are efficiently expressed when Salmonella replicates inside the macrophage vacuole.72 In line with this, both transcriptional and protein expression studies indicate that PgtE is induced and becomes functional during growth of Salmonella inside the macrophage vacuole.14,51,73 Inside HeLa epithelial cells, transcription of pgtE is more efficient than in culture media, but weaker than inside murine macrophages.73 Thus, PgtE seems to be strongly associated to the interaction of Salmonella with the macrophage.

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Haiko, Suomalainen, Ojala, La¨hteenma¨ki, Korhonen A Plasminogen

B

0h 1h 2h 5h 10h 22h

1.4 Δ A405 nm/30min

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tE Pg

pT m O

E3

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Omptins show remarkable variability in proteolysis of biologically important polypeptides, as illustrated in Figure 3, where the reactivities with plasminogen, gelatin, and matrix metalloproteinase-9 (MMP-9) are shown for OmpT, Pla, and PgtE expressed in recombinant E. coli.14,16,20,21 Below, the three omptins are compared in relation to their reported substrates and functions.

tE

pT m O

1.8 1.6

ATTACK ON INNATE IMMUNITY BY THE OMPT, PLA AND PGTE OMPTINS

Pg

Pl a

80

PlasminH Angiostatin

E3

Protease OmpT is active under extreme denaturating conditions and cleaves denaturated proteins.74 In keeping with this, its expression is increased in stress conditions, i.e. upon amino acid starvation or in the presence of antibiotics.75,76

pS

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C

Generation of plasmin D

4.0 3.5 Fluorescence (x105)

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m O

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pT

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E3

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Pg tE

m pT O

Pl

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proMMP-9 active MMP-9

pS E3 80

Protease Pla of Y. pestis is an efficient plasminogen activator (Fig. 3A,B). The generated plasmin is a powerful serine protease with several important physiological functions, which include degradation of fibrin clots as well as proteolytic activation of proMMPs.77,78 Once activated, the MMPs cleave collagen networks in extracellular matrices (ECMs) that form tissue barriers hindering cell migration.79 Consequently, the high level of active plasmin formed by Pla (Fig. 3A,B) is associated with efficient fibrinolysis (Fig. 3C) and plasminmediated cleavage of human pro-matrix metalloproteinase 9 (proMMP-9, pro-gelatinase B; Fig. 3E). Plasmin also degrades laminin, a major glycoprotein of basement membranes, which also serves as an adhesion target for Pla.80 The combined activities, adherence of Plaexpressing bacteria onto laminin/basement membrane and simultaneous generation of plasmin by Pla, lead to rapid and uncontrolled in vitro degradation of laminin and ECM from human lung epithelial cells.2,80 Thus, the localized generation of uncontrolled plasmin activity at ECMs and basement membranes enables a powerful attack by Y. pestis on tissue barriers to migrate from subdermal to lymphatic tissue. This is mechanistically reminiscent of the localized proteolytic attack on tissue barriers by metastatic tumor cells, which led us to the definition of ‘bacterial metastasis’ to separate the process from bacterial invasion into cells.2 The role of the efficient plasminogen activation in the pathogenesis of bubonic plague is underlined by the finding that plasminogen-deficient mice show a 100-fold increase in resistance to Y. pestis infection.81 Plasmin activity is tightly controlled both at the levels of activation and proteolytic activity. Free plasmin activity

Fig. 3. Functional diversity of Pla of Y. pestis, OmpT of E. coli, and PgtE of S. enterica expressed in recombinant E. coli. (A) Plasminogen cleavage by the three omptins. Plasminogen was incubated for 24 h at 37 C with the bacteria, and plasminogen degradation was assessed by Western blotting with anti-plasminogen antibodies. Migration of plasminogen, plasmin heavy chains (PlasminH), and angiostatins are shown on the left. (B) Formation of active plasmin by the bacteria. Plasminogen was incubated with the bacteria for the time intervals indicated on the right, and plasmin activity was then measured for 30 min. (C) Degradation of fibrin. The bacteria (107 cells) were applied on the fibrin plate, and dissolution of the fibrin clot was visualized after incubation for 20 h at 37 C. (D) Degradation of DQ-gelatin by the bacteria. The DQ-gelatin was incubated with the bacteria for 5 h at 37 C, and the released fluorescence was then estimated. (E) Activation of proMMP-9 from human macrophages by the bacteria. Conditioned medium from human U-937 macrophages was incubated with the recombinant bacteria in the presence or absence of plasminogen, and samples of incubation supernatants were analyzed by gelatin zymography. pSE380 indicates the E. coli strain with the vector plasmid. Note that only Pla creates high plasmin activity and fibrinolysis and that only PgtE cleaves DQ-gelatin and proMMP-9 in the absence of exogenous plasminogen.

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Attack on innate immune defences by omptin surface proteases of enterobacterial pathogens 73 is rapidly inactivated by the circulating antiprotease a2-antiplasmin (a2AP).82 Thus Y. pestis creates uncontrolled plasmin activity simply by proteolytically cleaving and activating plasminogen and inactivating a2AP.20 Both of these functions are mediated by Pla.20 Some other bacterial pathogens overcome the control system by immobilizing plasmin/plasminogen on their cell surface, where plasmin is protected from binding by a2AP and thus functional.2,5 Plasmin is a key player in the control of the balance in the human homeostatic coagulation/fibrinolysis system.83 Fibrin clots are formed during coagulation at injured micro- and macrovascular blood vessel walls as one of the consequences of inflammation, and fibrin deposition is considered an important means of immune control to inhibit spread of bacterial pathogens.4 Degradation of fibrin clots is a major physiological function of plasmin (see Fig. 3C). In pneumonic plague, infection by a Pla-expressing strain of Y. pestis was associated with fibrin deposition altered from that observed in infection by a Pla-negative strain.12 Similarly, in intravenously infected mice, a reduced fibrin(ogen) deposition in liver lesions was detected around foci of Pla-expressing bacteria, as compared to the foci observed with Pla-negative Y. pestis.84 No difference was evident in plasminogen-free mice. Interestingly, the lesions with Pla-expressing bacteria showed almost complete absence of inflammatory infiltrates, which, in contrast, were observed in hepatic lesions in animals infected with Pla-negative bacteria.84 A similar difference in the number and distribution of inflammatory cells, predominantly polymorphonuclear neutrophils, had been earlier observed in murine subcutaneous lesions after injection with Pla-positive and Pla-negative Y. pestis.11 These observations were interpreted to be in line with the observations that the aMb2 integrin-mediated engagement of fibrin(ogen) on leukocyte surface is critical for leukocyte function and innate immunity in vivo.84,85 Loss of fibrin(ogen)– integrin interaction leads to an impediment in bacterial clearance and in ability of leukocytes to implement antimicrobial functions fully.85 These findings indicate that plasmin-mediated fibrinolysis is a major pathogenetic function of Pla which enhances bacterial migration and possibly also regulates inflammatory responses. A recent study indicated that the interaction of Pla with the coagulation/fibrinolysis systems is complex. Protease Pla was found to cleave the anticoagulant tissue factor pathway inhibitor (TFPI) and to counteract TFPImediated inhibition of clot formation.86 This would enhance coagulation, and it is interesting that OmpT was equally active whereas PgtE was 100-fold less active in TFPI inhibition. The authors made the hypothesis that Y. pestis promotes activation of clotting system to form a protective fibrin barrier against host inflammatory cells

at early stages of infection.86 Clearly, interaction of Pla and other omptins with the coagulation/fibrinolysis systems is a topic for future studies. The omptins PgtE and OmpT are poor plasminogen activators and are not able to cause significant fibrinolysis (Fig. 3C). In particular, PgtE cleaves plasminogen efficiently (Fig. 3A) but is a poor activator (Fig. 3B), which indicates that it cleaves to inactivate the plasminogen molecule. Our on-going work has shown that similar poor activation is also shown by the omptins detected in Legionella pneumophila and Erwinia pyrifoliae.87,88 Salmonella PgtE efficiently inactivates a2AP.14 In Salmonella pathogenesis, it seems that inactivation of a2AP is more critical than generation of plasmin. The macrophage host cell of Salmonella secretes the mammalian plasminogen activator urokinase89 and this probably ensures enough plasminogen activator activity in the vicinity of Salmonella cells. Urokinase-mediated conversion of plasminogen to plasmin on the macrophage surface is a self-amplified system, as plasmin, in turn, activates pro-urokinase.89 Antiprotease a2AP delays plasminogen activation in lysates of uninfected murine macrophage-like J774A.1 cells, whereas lysates of Salmonella-infected cells are resistant to inhibition by the antiprotease. This effect is also mediated by PgtE, as lysates of cells infected with a pgtE-deficient derivative of Typhimurium are as sensitive to a2AP as uninfected cells.14 Thus, although PgtE activates plasminogen poorly, it appears to promote both the generation and the maintenance of plasmin activity by taking advantage of host cell functions and by destroying the control system for plasmin. Activation of MMP and gelatin degradation Protease PgtE of S. enterica is the only omptin so far found to activate the macrophage-derived proMMP-9 in the absence of exogenous plasminogen (Fig. 3E). Further, PgtE also degrades gelatin, whereas Pla and OmpT are weak in this function (Fig. 3D). Different MMPs promote migration of mammalian cells through ECM and are also important in inflammatory reactions by cleaving cytokines, chemokines and antimicrobial peptides.79,90 Like plasminogen, MMPs are secreted in a pro-enzyme form and require proteolytic cleavage for activation. Inactive proMMP-9 is secreted by cells of the monocyte/macrophage lineage, and its secretion is strongly enhanced by bacterial surface structures such as LPS.90 Thus, Salmonella both stimulates the production of the pro-enzyme and proteolytically converts it to an active enzyme. Mature MMP-9 efficiently degrades gelatin, and PgtE thus promotes gelatin breakdown in both direct and indirect manners.16 In addition to gelatin,

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which is a natural degradation and denaturation product of type I collagen, MMP-9 degrades type IV collagen and various other ECM components. These activities are likely to enhance migration of Salmonella and Salmonella-infected host cells through ECM. The possible interactions of PgtE with other MMPs require further studies. It has been reported that MMP-9 deficient mice are more resistant to S. enterica-caused enterocolitis91 and MMP-3 deficient mice to systemic salmonellosis92 than wild-type mice. These results indicate that MMPs have a role in Salmonella infections. It is noteworthy that PgtE resembles mammalian MMPs in its substrate specificity. Enzyme MMP-3 degrades gelatin, activates proMMP-9, and inactivates a2AP,93,94 which are all functions of PgtE as well. Like the other omptins, PgtE has a catalytic mechanism different from the zinc-dependent MMPs, and has only limited, local sequence similarity with MMP-3.16 However, PgtE seems to functionally mimic mammalian MMPs. Complement inactivation The observed serum resistance of Salmonella derives from cleavage of complement components by PgtE.15 The human serum complement system is a major component of innate immunity and confers resistance to pathogenic bacteria. Protease PgtE cleaves the complement proteins C3b, C4b, and, less efficiently, C5, to smaller molecular mass cleavage products.15 Complement proteins C3b and C4b, which are the circulating products of C3 and C4, are components of convertases that are critical in the activation of the complement cascade. During complement activation, C5 is cleaved to C5a, which attracts phagocytes to the infection site, and to C5b which is part of the membrane attack complex causing lysis of the cell wall in Gramnegative bacteria.95 Thus, proteolytic inactivation of C3b, C4b, and C5 is likely to increase the survival of Salmonella in the host. The O-antigen is the major factor that confers serum resistance of Salmonella, and patient isolates of Typhimurium invariably have a long O-antigen chain after growth in laboratory media.43 However, shortening of the O-chains in Typhimurium cells during growth within the macrophage vacuole described above14 suggests that other resistance mechanisms to complement are required at the time of bacterial release from dying macrophages. The high activity of PgtE in Salmonella released from infected macrophages suggests that PgtE-mediated complement protein inactivation is important under circumstances when the protective effect of LPS O-antigen is absent. The plague bacterium Y. pestis Pla has been found to cleave the complement protein C3, and it thus suppresses migration of inflammatory cells at the infection site.11

Degradation of C3 might also reduce opsonophagocytosis of Pla-expressing bacteria via the C3b receptors and thus promote the serum resistance of Y. pestis. Overall, the possible pathogenetic role of complement protein degradation by Pla has not been analyzed in detail. It, however, seems not to be a major virulence mechanism for Pla, as Y. pestis strains lacking Pla remain serum resistant due, for example, to immobilization of the complement regulator C4b-binding protein on bacterial surface.11,96 Interaction with host cells Histological studies on plague pathogenesis have repeatedly revealed the presence of extracellular, free bacterial cells at infection foci, which has led to the conclusion that plague is predominantly an extracellular infection.10,84,97–99 On the other hand, several reports have shown that Y. pestis enters, and is able to multiply in, mammalian epithelial and phagocytic cells in vitro and in vivo.100–106 The ability to multiply within macrophages is shared by Y. pseudotuberculosis, from which Y. pestis has evolved,27 and is dependent on the Yersinia virulence plasmid (pYV) that encodes a type III secretion system and a set of pathogenicity factors conferring resistance to phagocytic killing of yersiniae.107 Omptin Pla has been implicated as a bacterial ligand binding to, and mediating invasion of, Y. pestis into human epithelial cells as well as naı¨ve mouse macrophages.104,108 Recombinant E. coli expressing Pla induced actin re-arrangement in HeLa cells and the invasion was diminished in the presence of drugs that interfere with host cell signal transduction.109 A recent study reported that Pla is a ligand for the CD205 molecule on mouse macrophages and that blocking of this interaction reduces the dissemination of Y. pestis from the intranasal infection site into secondary organs.108 The studies above show that Y. pestis invades and multiplies within mammalian epithelial and phagocytic cells and that Pla has a role in bacterial uptake into the cells. It remains to be established whether the replication in phagocytes is a transient event in plague pathogenesis or whether (a fraction of the) bacteria disseminate to secondary infection sites within phagocytic cells. Unlike Pla, Salmonella PgtE does not mediate invasion into human epithelial cells.110 However, PgtE promotes survival of Salmonella inside murine macrophage-like J774A.1 cells,14 which again emphasizes the association of PgtE with the macrophage host cell. Salmonella is known to migrate in the host by traveling within CD18-positive phagocytic cells.13 Kinetics of mouse salmonellosis after intraperitoneal infection suggests that PgtE may promote cell migration: a significant

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Attack on innate immune defences by omptin surface proteases of enterobacterial pathogens 75 reduction in the organ load was seen with the pgtEdeficient derivative 1 day post-infection,16 i.e. at the time when salmonellae have been transported to the liver and the spleen by phagocytes that ingest bacteria in the peritoneal cavity. After longer infection times, numbers of the pgtE-deficient derivative remained lower but increased at the same rate as numbers of the wild-type bacteria.16 This suggests that PgtE is required at the stage of the infection when cell migration is prominent and proteolysis is needed. However, although Salmonella is known to generate ample and variable PgtE-dependent proteolysis in cell models, the specific roles of PgtEderived proteolytic activities in Salmonella infections remain to be established in vivo. Very little is presently known about possible induction of cytokine secretion by omptins. Brandenburg and co-workers111 tested the effect of OmpT protein and OmpT–LPS mixtures to induce the production of TNF-a in human mononuclear cells. Increased production was observed in the presence of OmpT. Use of Chinese hamster ovary reporter cell lines indicated no involvement of TLR2 or TLR4 in the observed stimulation, and the authors concluded that the response was not due to LPS present in the OmpT preparation. Inactivation of antimicrobial peptides The growth advantage that the PgtE-expressing bacteria gain inside murine macrophages is likely to be due to the cleavage and inactivation of antimicrobial peptides. Protease PgtE cleaves antimicrobial peptides of both human (C18G, LL-37) and mouse (CRAMP) origin.112 Interestingly, antimicrobial peptides have been shown to act as activating signals for PhoP,113 which means that the expression of PgtE is enhanced in the presence of its substrates. Antimicrobial peptides are present inside the macrophage vacuole and also secreted in large quantities by Paneth cells in the intestinal epithelium.114 In Paneth cells, the secretion of a-defensins is triggered by LPS of Salmonella and other bacteria.114,115 However, it is not known whether the defensins in the intestinal lumen promote PgtE activity in Salmonella. In addition to direct bactericidal effects, antimicrobial peptides such as the cathelicidin LL-37 act as signaling molecules in, for example, chemotaxis of inflammatory cells.116 PgtEmediated proteolysis of antimicrobial peptides and its regulation can thus have far-reaching effects in crucial elements of the innate immunity system. Cleavage of antimicrobial peptides is a function shared also by Pla and OmpT. Pla was recently shown to cleave LL-37.117 Cleavage of LL-37 by Pla was prevented by the presence of capsular antigen fraction 1 on Y. pestis cells. This, however, had only marginal effect on plasminogen activation, suggesting substrate

specificity in the inhibitions. Protease OmpT degrades protamine, a cationic peptide secreted by uroepithelial cells118 and thus may enhance colonization of E. coli at uroepithelia.118

CONCLUSIONS There are several ways how Pla of Y. pestis and PgtE of S. enterica enhance bacterial resistance to innate immune defences and promote bacterial spread. Salmonella PgtE enhances cell migration directly, by attacking gelatinous components of the ECM, as well as indirectly, by enhancing host plasmin and MMP activities. Protease Pla enables the indirect mechanism, i.e. plasminogen activation. They both inactivate complement proteins and enhance either survival or uptake of bacteria inside host cells, as summarized in Figure 4. It is noteworthy that OmpT of E. coli is inefficient in the activities shown in Figure 3, which is in agreement with E. coli being a less invasive species than Y. pestis or S. enterica. It is remarkable how the functions of Pla and PgtE have evolved to support the life-style of their host bacterium, i.e. the predominantly extracellular pathogen Y. pestis and the intracellular S. enterica. The two omptins enhance surface proteolysis through variations of the same main theme – engagement of host-derived proteolysis (Fig. 4). The plasminogen/plasmin, the MMP and the complement proteolytic systems interact with each other and also influence and control cytokines and chemokines as well as leukocyte functions.90 This underlines that omptin proteolysis is not targeted to degradation of ECM components and/or fibrin alone but novel regulatory functions as well as biologically important proteolysis targets should be pursued. The functional differences that have been identified so far appear to result from minor sequence changes at the loop structures of omptins. In an attempt to mimic functional adaptation and molecular evolution of omptins, Pla has been modified into a PgtE-like gelatinase and OmpT into a Pla-like plasminogen activator by amino acid substitutions at L1–L5 regions (Fig. 2).16,20 The conclusion from this work is that the polypeptide substrate specificity of omptins is dictated by the flexible surface loops surrounding the conserved catalytic groove. The predicted amino acid sequences of Pla and PgtE are highly conserved within the bacterial species, whereas the OmpT sequences from different E. coli isolates show variation and fall into two major subgroups.6 This suggests that OmpT proteins in E. coli strains may express functional heterogeneity. Overall, the omptin protease family gives a prime example of an evolvable, robust enzyme fold that easily acquires novel or improved functions through amino acid substitutions at critical protein sites.119 The functional adaptation also

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Plasmin

MMP-9

ProMMP-9

α2AP INACTIVATION



COMPLEMENT C3b, C4b, C5 INACTIVATION

ECM DEGRADATION

Plasmin



Serum resistance

BREAKDOWN OF GELATIN

MMP-9

ProMMP-9 ACTIVATION

INACTIVATION OF ANTIMICROBIAL PEPTIDES

PLASMINOGEN ACTIVATION

Salmonella enterica PgtE

INACTIVATION OF ANTIMICROBIAL PEPTIDES

CLEAVAGE OF BACTERIAL CELL SURFACE PROTEINS

Escherichia coli OmpT

Fig. 4. Major pathogenetic functions identified for Pla, PgtE and OmpT. Pla is a powerful plasminogen activator and also releases the control by inactivating a2AP. The uncontrolled plasmin degrades fibrin clots, activates proMMPs that attack on subepithelial ECM, and also directly degrades laminin of basement membranes. Pla mediates invasion of Y. pestis into epithelial and phagocytic cells. Pla also degrades the complement protein C3; in plague, the main virulence function of C3 degradation is probably the inhibition of neutrophil recruitment at the infection site. PgtE is up-regulated and enhances growth of S. enterica in mouse macrophages by degrading antimicrobial peptides. PgtE is powerful in a2AP inactivation but a poor plasminogen activator; it can, however, utilize urokinase-mediated plasmin formation by activated macrophages. PgtE and plasmin activate proMMP-9, and plasmin degrades laminin. PgtE also degrades several complement proteins and increases serum resistance, which can be important upon release of salmonellae from macrophages. OmpT cleaves bacterial surface proteins and can also enhance bacterial colonization by degrading antimicrobial cationic peptides.

Inhibition of neutrophil recruitment

ECM DEGRADATION

COMPLEMENT C3 INACTIVATION

HOST CELL INVASION

ADHESION TO ECM LAMININ

FIBRINOLYSIS

α2AP INACTIVATION

PLASMINOGEN ACTIVATION

Yersinia pestis Pla

76 Haiko, Suomalainen, Ojala, La¨hteenma¨ki, Korhonen

Attack on innate immune defences by omptin surface proteases of enterobacterial pathogens 77 involves the control of the omptin expression which is different in the three omptins here described. Powerful surface proteolysis in Y. pestis and S. enterica is not solely dependent on the omptin molecules and their expression but is created through control of LPS structure which has a strong effect on omptin function.

17. 18.

19.

ACKNOWLEDGEMENTS 20.

This work has been financially supported by the European Union Network of Excellence EuroPathoGenomics programme, the Viikki Graduate School in Molecular Biosciences, as well as the Academy of Finland (grant number 116507).

21.

22.

23.

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