Relevance in pathogenesis research

Veterinary Microbiology 153 (2011) 2–12

Contents lists available at ScienceDirect

Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic

Review

Relevance in pathogenesis research Carlton L. Gyles * Department of Pathobiology, University of Guelph, Guelph, Ontario, N1G 2W1 Canada

A R T I C L E I N F O

A B S T R A C T

Article history: Received 23 December 2010 Received in revised form 9 April 2011 Accepted 13 April 2011

Research on pathogenesis of bacterial diseases involves exploration of the intricate and complex interactions among pathogen, host, and environment. Host–parasite–environment interactions that were relatively simple were the first to be understood. They include intoxications in which ingestion of a powerful bacterial toxin was sufficient to cause disease. In more complex cases bacteria occupy a variety of niches in the host and attack at an opportune time. Some bacterial pathogens have a brief encounter with the host; others are long-term guests. This variety of relationships involves a wide range of strategies for survival and transmission of bacterial pathogens. Molecular genetics, genomics and proteomics have facilitated understanding of the pathogens and hosts. Massive information often results from such studies and determining the relevance of the data is frequently a challenge. In vitro studies often attempt to simulate one or two critical aspects of the environment, such as temperature, pH, and iron concentration, that may provide clues as to what goes on in the host. These studies sometimes identify critical bacterial virulence factors but regulation of bacterial virulence and host response is complex and often not well understood. Pathogenesis is a process of continuous change in which timing and degree of gene expression are critical and are highly regulated by the environment. It is impossible to get the full picture without the use of natural or experimental infections, although experimental infections involve ethical and economic considerations which may act as a deterrent. ß 2011 Elsevier B.V. All rights reserved.

Keywords: Pathogenesis Research host–parasite interactions Environment Methodologies Infection

Contents 1. 2.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Recurring themes in pathogenesis . . . . . . . . . . . . Adherence . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Invasion . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Toxin production . . . . . . . . . . . . . . . . . . . . 2.3. Iron acquisition . . . . . . . . . . . . . . . . . . . . . 2.4. Mimicry . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Attacking or evading the immune system 2.6. 2.7. Multifunctional bacterial proteins . . . . . . . Protein secretion . . . . . . . . . . . . . . . . . . . . 2.8. Methodologies and context . . . . . . . . . . . . . . . . . . Nucleic acid-based methodologies . . . . . . 3.1. Proteomic and metabolomic studies . . . . . 3.2. Bioimaging . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.

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* Tel.: +1 519 824 4120; fax: +1 519 824 5930. E-mail addresses: [email protected], [email protected]. 0378-1135/$ – see front matter ß 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2011.04.020

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4.

3.4. Cell culture systems . . . . . . . Studies with organ systems . 3.5. 3.6. In vivo systems . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . .

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1. Introduction Pathogenesis research seeks to understand the intricacies of interactions among bacteria and host animals and the effects of environment on these interactions. It is also of considerable practical importance as it can lead to innovative diagnosis, treatment, prevention and eradication of disease of animals and humans. The first part of this review will address the issue of complexity at every step of pathogenesis. The subsequent parts will discuss the significance of recurring themes in pathogenesis and the importance of methodology and context for pathogenesis research. Pathogenesis is marked by complexities at every step, making it sometimes difficult to ascribe relevance to research findings. Recurring themes are often useful guideposts, as familiar patterns and parallels in other organisms frequently help to indicate the right direction. New methodologies are a major driver in pathogenesis research and access to a variety of tools has enhanced our ability to investigate pathogenesis, especially at the cellular and molecular levels. The context in which our studies are conducted is of extreme importance and needs to be carefully considered at both the study design and interpretation stages. Complexity in pathogenesis research is in part generated by the need to concern ourselves with not only the pathogen but also the host and the environment. Many researchers in pathogenesis research focus on the pathogen and start with identification of virulence and fitness genes, ascribing function to genes and proteins, and trying to understand regulation. Increasingly we have come to recognize the profound role of the host and the environment in regulation and function in bacterial pathogens. We have also come to recognize the added complexity associated with multifunctionality of some proteins, redundancy, co-operation, and the sequential changes that bacteria undergo on their pathogenesis journey. Pathogen, host and environment are all highly variable and all change as the process unfolds. Superimposed on this is the massive amount of genetic information which often has to be considered as advanced sequencing methodologies make it easier to generate sequence data. We often deal with the complexities by generating big picture concepts then hiving off aspects of these and pursuing them, particularly at the cellular and molecular levels. For example, major damage to the host is often compartmentalized as occurring primarily along a bacteria-mediated pathway, through the action of bacterial toxins, or primarily through a host-response pathway, which may or may not be toxin-mediated. Furthermore, substantial host-mediated damage occurs in infections such as Gram-negative bacterial sepsis, superantigenmediated diseases and tuberculosis – diseases that are

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largely the result of toxic substances released by host cells (macrophages, lymphocytes, neutrophils) in response to infection. In Gram-negative sepsis due to the Enterobacteriaceae, LPS from the cell wall is bound by the pattern-recognition molecule TLR4 in conjunction with the cell-surface receptor CD14. The binding of LPS leads to recruitment of the adaptor proteins MyD88 and IRAK to the cytoplasmic domain of TLR4. This complex initiates a signaling cascade of phosphorylation events ending with the release of NF-kB, which migrates to the nucleus where it activates the transcription of proinflammatory genes. Similar signal transduction pathways are activated by Gram-positive cell wall constituents such as peptidoglycan and lipoteichoic acid via TLR2 or TLR6. Interestingly, a number of oral Gram-negative bacteria, including Prevotella oris and Porphyromonas gingivalis interact with host cells through TLR-2, although some reports indicate that P. gingivalis can activate host cell pathways through both TLR-2 and TLR-4 (Bainbridge and Darveau, 2001; Nemoto et al., 2006; Konopka et al., 2010). The role of LPS in Gram-negative sepsis is clear; LPS also contributes to pathogenesis in other disease syndromes in ways that are more subtle and a potential role for LPS needs to be considered in pathogenesis of a wide variety of Gram-negative pathogens. A bacterial function may benefit both the pathogen and the host. One researcher mused as to whether granuloma formation represented confinement in the penitentiary or living in the penthouse condo (Paige and Bishai, 2010). It may well be that it is a bit of both. This is illustrated by studies with Rv0386, a Mycobacterium tuberculosis adenylate cyclase that subverts host-cell signal transduction, leading to a progranulomatous response with excess TNFalpha secretion. Loss of adenylate cyclase is associated with reduced TNF-alpha levels in mouse lungs and poorer bacterial survival (Agarwal et al., 2009). TNF-alpha is considered to be required for host containment of tuberculosis, but this study indicates that eliciting an excessive TNF-alpha response may be part of the bacterial virulence strategy. These observations suggest that the granuloma response may be advantageous to the pathogen; supporting this is data that bacterial mutants that lack granuloma promoting genes have reduced survival. This response is also beneficial to the host as it contains the pathogen, although allowing for the possibility of activation at a later time. Bacterial superantigens, potent lethal toxins associated primarily with Staphylococcus aureus and Streptococcus pyogenes, are examples of bacterial products that induce damage by the host reaction (Lappin and Ferguson, 2009; Stow et al., 2010). Superantigens trigger an excessive cellular immune response that can lead to lethal toxic shock. Bypassing the restricted presentation of conventional

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antigens, superantigens bind directly to most major histocompatibility (MHC) class II molecules and stimulate almost all T cells bearing particular domains in the variable portion of the b chain (Vb) of the T-cell receptor (TCR), without need for processing by antigen-presenting cells. Toxicity results from massive induction of cytokines derived from T-helper-type-1 (Th1) cells, which include interleukin (IL)-2, gamma interferon (IFN-g) and tumor necrosis factor (TNF)-b. Although the role of superantigens in human disease has been extensively investigated (Lappin and Ferguson, 2009), little is known about the role of superantigens in disease in animals. A 1987 paper (Almazan et al., 1987) reported that 25/66 Staphylococcus intermedius and 5/10 S. aureus from infections in dogs were superantigen-positive and a 2002 preliminary report on frequency of enterotoxins from S. intermedius found that 25/96 isolates had superantigens (Hendricks et al., 2002). Streptococcus canis causes toxic shock syndrome and necrotizing fasciitis in dogs (Miller et al., 1996) – conditions that seem to beg for superantigen involvement. Prescott and colleagues (DeWinter et al., 1999) failed to detect SPE A, B, C or SSA in 15 isolates from this condition but later identified an open reading frame (ORF) in a bacteriophage from S. canis that encoded a putative protein with 27% amino acid identity with pokeweed mitogen (Ingrey et al., 2003). This was particularly interesting because the phage could be induced by exposure to fluoroquinolones, a class of antibiotics associated with the disease. Thus, the role if any of superantigens in animal diseases has not been established. Examination of genomic data could facilitate further study of this topic.

Our research often fails to consider that bacteria frequently grow in multicellular communities that can exhibit complex phenotypes. A wide range of bacterial pathogens form biofilms in vitro and biofilms are implicated in over 60% of human bacterial infectious diseases. Some biofilms are multispecies. Oral biofilms comprise, in total, about 1000 species, only half of which are culturable. Biofilms have not been studied extensively in animal infections although many animal pathogens produce biofilms in vitro (Jacques et al., 2010). Only a few animal pathogens have been shown to form biofilms in vivo; these include Bordetella bronchiseptica binding to the nasal epithelium of infected mice (Sloan et al., 2007). Enterohemorrhagic E. coli (EHEC) and EPEC grown in shaking cultures secrete the effector proteins EspC and EspP, respectively, that oligomerize to form large macromolecular structures that exhibit adhesive and cytopathic properties and might act as a substratum for biofilm formation (Xicohtencatl-Cortes et al., 2010). However, we do not know whether this happens in vivo. Biofilm formation may play a major role in pathogenesis. For example, the lesion in staphylococcal endocarditis is a complex biofilm composed of bacterial and host components located on a cardiac valve (Parsek and Singh, 2003). This vegetation impairs valve function, is a source for prolonged infection of the bloodstream, and can form emboli that migrate to the brain, kidney, and extremities. Although the organisms are usually antibiotic-susceptible prolonged intravenous antibiotics and replacement of the infected valve may be required to treat the infection.

2. Recurring themes in pathogenesis

Several bacterial species are clearly invasive pathogens that penetrate epithelial or endothelial barriers and access underlying tissues (Dunn and Valdivia, 2010; Hartlova et al., 2010; Hunstad and Justice, 2010; Join-Lambert et al., 2010; Kim et al., 2010). There are others for which the significance of invasion is unclear. O157 EHEC, for example, invades some cultured cells (HCT, human colonic epithelial cells; RPMI, MAC-T) but fails to invade others (CHO, Chinese hamster ovary cells; HeLa) (Matthews et al., 1997; Luck et al., 2005). These bacteria also invade calf intestinal epithelial cells in vivo (Sandhu and Gyles, 2002). However, accounts of EHEC O157 pathogenesis do not usually consider a role for enterocyte invasion. In atypical EPEC a rare subset has been described that actively invade non-phagocytic host cells by inducing formation of membrane ruffles (Bulgin et al., 2009). In CaCO2 cells the EPEC strain uses EspT to induce ruffles, replicates in the vacuole and induces formation of intracellular actin pedestals around the circumference of the vacuole. This subset may be associated with hypervirulence. In contrast with what occurs with EHEC and EPEC, Salmonella Typhimurium is highly invasive in calves and invades the epithelium of the ileum, with greatest intensity in the dome region (Santos et al., 2002). Large numbers of bacteria are consistently seen in vacuoles in phagocytic cells. Interaction of the bacteria with the vacuole is not surprisingly a major focus of research on pathogenesis in this species.

Amidst the complexity, themes that recur in a wide range of host–parasite interactions are helpful. They identify patterns of behaviour that allow researchers to focus on specific genes, gene clusters, or proteins. Some of these themes are very common, others less so. Adherence, invasion, and toxin production are three features that are encountered at high frequency in a wide range of bacterial pathogens. 2.1. Adherence Adherence is a recurring theme, allowing bacteria to occupy a site or to use an initial attachment site as a launching pad into tissue (Le Bougue´nec, 2005; Speziale et al., 2009; Moxley and Smith, 2010). Some relatively simple patterns involve fimbrial adhesins as in F4+ porcine enterotoxigenic Escherichia coli (ETEC) and afimbrial adhesins as in human atypical enteropathogenic E. coli (EPEC). Within a bacterial species, adherence mechanisms may vary markedly. In bovine ETEC adherence requires fimbriae but capsular polysaccharide also contributes. In O157:H7 Shiga toxin-producing E. coli (STEC) adherence is more complex, involving more than 1 type of pilus initially and later the outer membrane protein intimin, a translocated intimin receptor (TIR) produced by the bacteria, and two host receptors (beta-1 integrin and nucleolin).

2.2. Invasion

C.L. Gyles / Veterinary Microbiology 153 (2011) 2–12

Shigella is also highly invasive, but is typically contained locally at the level of the intestine (Ashida et al., 2009). Although Shigella have neither adherence factors nor flagella required for attaching to or accessing the intestinal epithelium, ingestion of as few as 10 bacteria can result in disease. Shigella confront a colonic epithelium which is well defended by physical and chemical means that include mucin, tight junctions, defensins, cathelicidins, lysozyme, RNase A, proteases and secretory phospholipase. Shigella counters by secreting over 50 effectors through a type III secretion system (T3SS). These effectors include proteins that dampen the immune response and others that induce membrane ruffling and uptake of the bacteria. Shigella are endocytosed by the apical membrane of M cells, transported to the endosomal compartment, and exocytosed at the basal membrane. The bacteria then invade the resident macrophages, disrupt the vacuolar membranes, and move freely in the cytoplasm. They multiply in this location, induce an inflammatory response, and cause the death of the macrophages. The bacteria that are released enter the basolateral surface of the neighbouring epithelial cells by macropinocytosis. The process of escape from the vacuole, multiplication in the cytoplasm and spread to adjacent cells is repeated (Sasakawa, 2010). 2.3. Toxin production Bacterial toxins were the first virulence factors to be discovered. It is not surprising that the earliest bacterial toxins were those in which the bacterial strategy involved Gram-positive bacteria producing a single powerful protein toxin that caused dramatic effects in the host. In some cases, as with foodborne botulism, the bacteria may take no part in the disease process other than depositing its toxin in a convenient food (Shapiro et al., 1998; Mahajan and Brubaker, 2007). This pattern dominated our thinking for decades – Gram-positive organisms produce powerful specific protein toxins and Gram-negative organisms produce nonspecific lipopolysaccharide (LPS). We have had to broaden our concept of bacterial toxin to include superantigens such as the S. aureus toxic shock syndrome toxin (TSST) and enterotoxins, as well as effector proteins delivered on the surface or into the cytosol of host cells, as occurs with EPEC, EHEC, Salmonella, Shigella, and Yersinia. We have come to recognize a broad range of toxins involving a variety of activities that interact with the host at numerous sites and can cause damage by a variety of mechanisms. For many of these toxins, including some that have been known for decades, it is not even known whether they play a role in disease. These include E. coli cytotoxic necrotizing factor (CNF), E. coli enteroaggregative heat stable enterotoxin (EAST-1), and Clostridium botulinum C2 and C3 toxins. The patterns become much more complex when there are several toxins, many body sites, and a range of diseases as occurs with S. aureus, Pseudomonas aeruginosa and Clostridium perfringens for example. In the case of S. aureus the armamentarium includes structural components such as capsule, peptidoglycan and protein A; numerous toxins including the superantigens TSST and enterotoxins, leukocidins, hemolysins, and exfoliative toxins; and

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enzymes such as catalase, coagulase, hyaluronidase, fibrinolysin, lipases, nucleases, penicillinases, phosphatases, and proteases. In addition to the impressive array of virulence factors, S. aureus may also give rise to small colony variants, which allow intracellular persistence, greater resistance to antibiotics, and chronicity (Tuchscherr et al., 2010). 2.4. Iron acquisition Almost all bacterial species require iron because it functions as a central component of molecules required for energy generation, oxygen transport, nucleotide biosynthesis, and protection against oxidative damage (Nairz et al., 2010; Skaar, 2010). Borrelia burgdorferi is a notable exception as it appears to require manganese, zinc and magnesium but not iron for growth in vitro and analyses of cell lysates, and its genome showed an almost complete absence of genes encoding iron-containing proteins (Posey and Gherardini, 2000). Elimination of the genes for most proteins that require iron as a cofactor and substitution of manganese for iron in the few metalloproteins that are produced is considered to be a novel strategy used by this pathogen to overcome the iron limitation imposed by its mammalian hosts. In the struggle between host and bacterial pathogen for iron, bacteria have developed a number of other strategies that respond to host mechanisms for withholding iron, referred to as nutritional immunity (Nairz et al., 2010; Skaar, 2010). Sequestration of iron is the major component of this nutritional immunity. Host iron is sequestered intracellularly as a part of complexes such as ferritin, hemoglobin and myoglobin. Extracellularly, iron is bound to the serum protein transferrin. Pathogenic bacteria have evolved to detect low iron availability as a marker of vertebrate tissue and have evolved a number of high-affinity iron uptake systems for retrieving iron from their mammalian hosts. These systems consist of siderophore-mediated iron capture, heme acquisition systems, and use of transferrin/lactoferrin receptors (Weinberg, 2009; Skaar, 2010). Iron or iron-containing compounds such as heme is captured by bacterial siderophores or surface proteins and transported through the cell membranes into the cytoplasm. Interestingly, Neisseria gonorrheae (a human pathogen), Mannheimia haemolytica (a bovine pathogen) and Actinobacillus pleuropneumoniae (a pig pathogen) each use transferrin only from their cognate host as an iron source. It appears that both host and bacteria have evolved over time to restrict iron and to extract iron, respectively. This is suggested by examination of iron utilization by Salmonella. One of the proteins upregulated in the host in response to Salmonella infection is an antimicrobial peptide lipocalein 2, which binds specifically to the bacterial siderophore enterobactin, preventing its use in bringing iron to the iron-starved Salmonella. Salmonella Typhimurium has countered by producing salmochelin which is not bound by lipocalein 2 (siderocalein). Salmochelin is found in Salmonella enterica, uropathogenic E. coli (UPEC) (Hantke et al., 2003), neonatal meningitis E. coli (NMEC), and avian pathogenic E. coli (APEC).

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2.5. Mimicry

2.7. Multifunctional bacterial proteins

Mimicry is sometimes used to circumvent host defence as indicated by the following examples. The sialic acid K1 capsule of NMEC in humans, or APEC in chickens and the sialic acid moiety of group B streptococcus contribute to virulence by interfering with host complement activation and opsonophagocytosis (Chaffin et al., 2005; Bliss and Silver, 1996; Kariyawasam et al., 2006; Goller and Seed, 2010). The Salmonella type III secreted effector proteins SptP and SopE modulate changes in the cytoskeleton of host cells by acting as functional mimics of molecules that regulate Rho family signaling proteins (Schlumberger and Hardt, 2005). Listeria ActA mimics the actin nucleation promoting factor WASP (Wiskott–Aldrich syndrome protein) and recruits the actin-nucleating protein ARP2/3 complex to the bacterial surface to facilitate propulsion by actin comet tails (Chong et al., 2009). In Yersinia pseudotuberculosis the integrin-binding region of the envelope protein invasin mimics the host integrin ligand, fibronectin and manipulates signal transduction pathways in the host thereby contributing to bacterial attachment and internalization (Hamburger et al., 1999).

Multifunctional proteins contribute to bacterial efficiency. For example, the numerous virulence factors of S. aureus include multifaceted proteins such as Protein A (von Ko¨ckritz-Blickwede, 2009). Protein A is primarily known for its ability to bind immunoglobulins at the Fc end making it inaccessible to opsonins, but it has other properties that modify biological responses. By binding to human von Willebrand factor (vWF), protein A facilitates the adherence of S. aureus to vWF-coated surfaces such as endovascular catheters. Protein A can also bind to a tumor necrosis factor receptor (TNFR-1) that is widely distributed on the airway epithelium and stimulate inflammation in the lung. This interaction plays a central role in the pathogenesis of staphylococcal pneumonia. Protein A can cripple humoral immunity by binding to the VH3 region on exposed IgM molecules, leading to proliferation and apoptosis, and failure to mount a robust immune response. Protein A also promotes bacterial aggregation and formation of biofilms. This is an exceptional virulence factor, a single protein that can target multiple immunologically important processes. It is probably not a coincidence that it is among the most highly conserved staphylococcal virulence factors, or that its level of expression is significantly increased in staphylococci isolated from invasive human infections.

2.6. Attacking or evading the immune system The leukotoxin of M. haemolytica, an RTX toxin, attacks immune system cells and is critical to pathogenesis of bovine pneumonic pasteurellosis. The effects of LKT are specific for ruminant species, due to their possession of a unique beta-2 integrin receptor on leukocytes (Thumbikat et al., 2005). At high concentration, LKT induces formation of transmembrane pores and subsequent cell necrosis. Leakage of leukocyte contents into the surrounding pulmonary parenchyma contributes to fibrinous and necrotizing lobar pneumonia. The effects of LKT are enhanced by lipopolysaccharide, which is associated with the release of proinflammatory cytokines from the leukocytes, activation of complement and the coagulation cascade, and cell cytolysis. The lethal toxin (LT) of Bacillus anthracis is a zincdependent protease that cleaves MAPKK and leads to death of macrophages and endothelial cells (Khan et al., 2010). This reduces the ability of the body to counter the invasion of B. anthracis cells. Certain bacterial proteins directly destroy host immunoglobulin. IgA1 proteases are autotransporter proteolytic enzymes that cleave specific peptide bonds in the human immunoglobulin A1 (IgA1) hinge region sequence (Parsons et al., 2004). Several species of pathogenic bacteria secrete IgA1 proteases at mucosal sites of infection to destroy the human IgA1 and inactivate a major component of host defence. They have been identified in a number of human pathogens including Haemophilus influenzae, Neisseria meningitidis, N. gonorrhoeae, and Streptococcus pneumoniae. The balance between IgA antibodies against surface antigens of the respective bacteria and their IgA1 protease is an important factor in pathogenesis. There are no published examples of IgA1 proteases by bacterial pathogens of animals.

2.8. Protein secretion Pathogenic bacteria secrete virulence factors by a variety of secretion pathways (Holland, 2010). The type III secretion (T3SS) pathway is of particular interest for its ability to deliver bacterial proteins on and in host cells. Salmonella, Shigella, Yersinia, EHEC, P. aeruginosa, and Burkholderia pseudomallei all use T3SS as a major component of pathogenesis. The T3SSs are intricately structured organic nanosyringes that achieve the translocation of bacterial proteins (effectors) from the prokaryotic cytoplasm across three membranes into the host cytosol. The effectors may be encoded by genes at various locations in the genome, are sometimes multifunctional, and lead to a wide range of host cell activities such as cytoskeletal remodelling, immune modulation, and hypersecretion. Some pathogens use a type IV secretion system (T4SS) to translocate DNA and protein across the bacterial cell membrane. This system can affect pathogenesis through the transfer of plasmids and integrative and conjugative elements (ICEs) between bacteria as well as by transfer of effector molecules into the cytosols of eukaryotic target cells (Alvarez-Martinez and Christie, 2009). The role of this system in pathogenesis has been investigated in several pathogens, notably Legionella pneumophila, Helicobacter pylori, Brucella species, Bordetella pertussis, Coxiella burnetii, and Bartonella species. It is estimated that over 80 effector proteins are transferred into the host cell cytoplasm by L. pneumophila (reviewed by Ensminger and Isberg, 2009). A wide range of virulence-related functions have been ascribed to the T4SS. It is essential for the formation of a replication vacuole, which requires remodelling of the endocytic pathway (Raychaudhury et al., 2009). In L.

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pneumophila-infected mice, wild type bacteria cause an increased production of proinflammatory cytokines, compared with bacteria in which the T4SS was inactivated (Shin et al., 2008). An increase in production of proinflammatory cytokines has also been associated with the T4SS-translocated CagA protein encoded by the H. pylori cytotoxin-associated (cag) pathogenicity island, and small molecules which block this protein reduced the virulence of the bacteria in a mouse model of infection (Hilleringmann et al., 2006). In Brucella species the VirB T4SS is necessary for survival in phagocytic cells and for persistence in the host. The host’s innate immune response to this protein reduces bacterial proliferation, resulting in a balance between host and pathogen that gives rise to prolonged infection (de Jong et al., 2010). In B. pertussis the ADP-ribosylating pertussis toxin, a critical virulence factor and protective antigen, is secreted by a T4SS, which is encoded by ptlA-I. The ptlA-I genes are adjacent to the genes that encode the toxin and both sets of genes are cotranscribed (Shrivastava and Miller, 2009). In C. burnetii, alkaline proteins secreted by the T4SS appear to play a major role in permitting this obligate intracellular parasite to reside in the acidic phagolysosome (Samoilis et al., 2010). 3. Methodologies and context The methods that are chosen profoundly affect the kinds of results that are obtained. Typically each method has both advantages and disadvantages and there is often benefit to selecting several methods for investigating a particular aspect of pathogenesis. 3.1. Nucleic acid-based methodologies Over the past decade there have been impressive advances in microbial functional genomics, resulting from developments in microarray technology and genome sequencing (Pallen and Wren, 2007; Beaume et al., 2010; van Vliet, 2010). The earlier studies largely relied on hybridization involving microarrays, which are limited in the range of transcript levels that can be detected but the advent of next generation sequencing has permitted detection at the single nucleotide level (van Vliet, 2010). Large-scale sequencing has ushered in the development of high-throughput genome-wide expression techniques such as DNA microarrays which permit genomic comparisons and the simultaneous examination of pathogen and host genomes. Analyses of single nucleotide polymorphisms (SNPs) are especially important for pathogens such as B. anthracis, Mycobacterium, Y. pestis that have very little apparent variability. Although there is not always a linear relationship between the expressed genes and the proteins synthesised, transcriptome studies give us a reasonable insight into the state of the cell. Chatterjee et al. (2006) used whole genome microarraybased expression of a wild type and a hlt/plc double mutant Listeria monocytogenes in macrophage and epithelial cell lines compared with bacteria grown in broth cultures. They collected the bacteria at various times. They found that 484 genes (17% of the total genome) were differentially

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regulated; 301 were upregulated and 182 were downregulated during intracellular growth. Sixty-six genes were specifically up-regulated for adaptation in the vacuolar compartment and 115 for growth in the cytosolic compartment. Joseph et al. (2006) also used whole genome DNA microarrays to determine the expression profiles of Listeria in epithelial cells. Nineteen percent of the genes were differentially expressed compared with bacteria grown in Brain Heart Infusion broth. They identified 282 up-regulated genes and 259 down-regulated genes and validated their findings by screening a random mutant library of L. monocytogenes for intracellular growth deficiencies. They showed that 36% of all the up-regulated genes were required for proliferation in the cytosol. It is challenging coping with the torrent of new data being generated by rapid less expensive sequencing; determining functional relevance to the sequence data, and integrating the information with information from other technologies will be important. 3.2. Proteomic and metabolomic studies Proteomics of infected tissues can be very informative (Bumann, 2010). The genome is determined exclusively by heredity, but the proteome arises from both heredity and environment. The metabolome characterizes the collection of low molecular weight components (<1500 Da) in a biological system that could be cells, tissue, fluid or whole organisms. Metabolomics refers to the global and nontargeted quantitative analysis of the metabolome. While transcriptomics and proteomics profile the gene transcription and protein translation, respectively, metabolomics examines the ultimate downstream effect of changes in transcription and translation. Therefore, metabolomics could potentially provide us with molecular events close to the phenotype(s) under investigation. A great challenge lies in the development of methods for metabolite measurement that are sufficiently general while still sensitive enough to detect the low abundant species and the subtle concentration changes involved in transition between various phenotypes (Vieites et al., 2009). Metatranscriptomics (community transcriptomics) seeks to identify the genes that are expressed by all species within a complex specimen such as a fecal sample (Chistoserdova, 2010). Metaproteomics similarly is the study of proteins collectively expressed within microbial communities (Wilmes and Bond, 2006). Inside the cell the bacteria must adapt to a specialized environment. Oxygen, pH, nutrients will vary between cytosolic and phagosomal compartments. Cytosolic bacteria have access to all the nutrients available to the cell. The metabolic potential and the putative central metabolic pathways can be deduced from genome sequences and tested experimentally. Recurring themes are a great help in sifting through the data from genome, transcriptome, and proteome analyses and determining the direction to pursue, bearing in mind that bacteria sometime use different evolutionary paths to achieve similar objectives. Massive amounts of bacterial genomic data continue to accumulate but translation into functional correlates lags behind. For example, recent metabolic studies on the obligately anaerobic Clostridium

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acetobutylicum, showed that while genome annotation suggests the absence of most tricarboxylic acid (TCA) cycle enzymes, the bacterium has a complete TCA cycle (Amador-Noguez et al., 2010), demonstrating the importance of complementing genome annotation with studies to determine function. 3.3. Bioimaging Bioimaging is a powerful tool in pathogenesis research. Whole animal studies allow researchers to visualize and follow infections with labelled bacteria in the host. Recently, fluorescence dilution was used to identify both bacterial killing and bacterial replication in the interaction between bacteria and macrophages (Helaine et al., 2010). These researchers developed a reporter system based on fluorescence dilution that allowed them to directly quantify the replication dynamics of Salmonella Typhimurium in murine macrophages. These studies determined that Salmonella pathogenicity island 2 T3SS was necessary for the bacteria to replicate but did not promote resistance to killing and that many bacteria in the macrophages did not replicate but entered what appeared to be a dormantlike state. Microfluidic platforms have been developed for realtime imaging of host–pathogen interactions and cell signaling events (James et al., 2009). Such a system has been used with macrophage-like cells to interrogate the kinetics and stochasticity of immune response to pathogenic challenges. 3.4. Cell culture systems Cell cultures are widely used for in vitro studies involving interaction of bacterial pathogens and selected cells, recognizing the absence of critical structures and functions that operate in vivo. Attempts are usually, but not always, made to match the pathogen with cells of the relevant host and site. Much has been learnt from these studies but interactions are often influenced by factors such as the type of cell and culture conditions for the cell lines. Polarized cells improve the orientation so that bacteria can interact with the relevant surface and that barrier functions can be examined. Three-dimensional versions of cell cultures are now available and enhance the capacity of such systems. An in vitro flow adhesion assay that mimics the conditions found within blood vessels was developed with immortalized human microvascular endothelial cells coated on glass slides mounted in a parallel plate flow chamber (Grubb et al., 2009). Interestingly, adherence data obtained from this model are sometimes quite different from the findings under static conditions. Microfluidic devices offer the possibility of working at single cell resolution with biofilms under physiological flow velocities and with precise control of environmental conditions. 3.5. Studies with organ systems Organ culture is a valuable system for investigation of interactions of bacterial pathogens with relevant host

tissues. This technique is a valuable step up from cell culture but its limitations need to be recognized. Most infections are initiated at the skin or a mucosal surface and the pH, nutrients, oxygen level, immune cells, presence of cilia, movement, unwelcoming flora are all important factors in pathogenesis. Organ systems typically have several cell types and different environments that may result in a variety of types of interactions with the pathogen. The gastrointestinal system, for example, is made up of remarkably different organs along the system and sequential exposure of bacteria to the different elements may play a significant role in pathogenesis. Specialized cells include parietal cells, goblet cells, M cells, enterochromaffin cells, Paneth cells, and intestinal epithelial cells. The M cells are specialized epithelial cells of mucosal surfaces and are important in sampling antigen and sometimes constitute a portal of entry of pathogens. Interestingly, it appears that bacteria in one area not only adapt to that area but also prepare themselves for the next experience. Host cellular receptors for bacteria and toxins are critical for adherence, internalization, and activation, but the presence of receptors may vary among individual animals. For the bacteria, receptors include a range of surface structures that are often the sugar portion of glycoconjugates or proteins. For toxins, specificity and internalization are often determined by surface receptors. In anthrax, for example, PA molecules bind the product of the host capillary morphogenesis gene 2 (CMG2), link to form a heptameric complex that binds LF or EF; the toxin– receptor complex becomes internalized, and subsequently the LF or EF exits through a pore formed by a pH responsive conformational change in PA (Young and Collier, 2007). Temperature, iron, pH and flora all affect pathogenesis in important ways but organ culture systems vary considerably in the extent to which they take these factors into account. Importantly, these organ culture systems are usually set at body temperature. In Shigella flexneri, for example, most of the virulence-related proteins are upregulated at 37 8C (Zhu et al., 2010). In E. coli, alphahemolysin, P pili, K88 (F4) fimbriae, 987P fimbriae, and S pili are all repressed below 37 8C. In Streptococcus suis, suilysin production is also temperature regulated. The iron concentration of the culture medium may affect bacterial growth and metabolism as bacteria have developed a range of systems which capture and internalize iron. Low pH, as occurs in the stomach is a major defence of the host gastrointestinal system and bacteria causing infections in the intestine go through this acid bath and whatever selection in quantity and quality it may impose. The potentially selective effect of this experience could affect interactions with the intestine. The flora at various sites plays a significant role in pathogenesis and is usually not a part of organ culture systems. The skin, like the gut, contains an abundant microbial flora that varies in composition depending upon location. Staphylococcus epidermidis is a consistent skin colonizer whose lipotechoic acid suppresses skin inflammation during wound repair, thereby preventing an excessive inflammatory response (Jones et al., 2005). S. epidermis also interferes with signaling by toll-like receptors

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(TLRs). Metagenomic studies are revolutionizing the amount of information on human gut bacteria. In a recent study by Qin et al. (2010) sequencing of DNA from fecal samples from 124 individuals led to a total microbial gene catalog containing 3.3 million non-redundant predicted coding sequences. The entire cohort harboured between 1000 and 1150 prevalent bacterial species and each individual had at least 160 such species, which are also largely shared. A large proportion of the genes were poorly characterized. There were 57 species that were common to 90% of the individuals but there was high variability in the abundance of these species between individuals. Nutrients can profoundly affect pathogenesis and need to be carefully considered in preparation of bacterial inocula and maintenance of organ cultures. There is complete abolition of invasion of HeLa cells by wild-type S. flexneri by ornithine, uracil, methionine or branchedchain amino acids (Durand and Bjo¨rk, 2009). The inhibitory effect of the nutritional environment is stronger than that provided by temperature. Preparedness for the anticipated temporal order of environmental change appears to occur in the response of E. coli to lactose in the upper small intestine. The first stimulus (lactose) induces a response to that stimulus and the upcoming stimulus (maltose) which is found lower in the intestinal tract. So E. coli exposed to lactose induces the lactose operon highly and the maltose operon to a lesser extent. The reverse does not happen and if the E. coli are grown for 500 generations in high levels of lactose without subsequent exposure to maltose, the adaptation is lost. Osmoadaptation is important in the virulence of many bacteria (Sleator and Hill, 2002) and care should be exercised in matching the osmolarity of the organ culture system with that of the pathogen’s habitat in the host. Osmolarity-dependent changes in T3SS synthesis in Shigella appear to be controlled at the post-transcriptional level, through the regulation of InvE synthesis. Survival of uropathogenic E. coli (UPEC) in the murine urinary tract is dependent on its ability to respond to osmotic stress through the action of OmpR, the response regulator part of the OmpR–EnvZ two-component regulatory system. Oxygen concentration is a significant factor in pathogenesis of many bacteria and is usually adjusted in organ culture systems. M. tuberculosis responds to low oxygen levels by entering into a metabolically altered state, from which it later reactivates (Yuan et al., 1996). In Vibrio cholerae oxygen plays an important role in virulence regulation but the combination with pH is important (Kovacikova et al., 2010). In EHEC, Shigella and Salmonella, the bacteria appear to produce effectors in the anaerobic conditions of the intestinal lumen, priming them for their association with the intestinal epithelium where the small zone of oxygenation provides a different signal (Baxt and Goldberg, 2010). In P. aeruginosa, production of both LPS and alginate is influenced by oxygen concentration (Schertzer et al., 2010). Secretions such as bile may moderate virulence. For example, bile salts activate the Shigella T3SS (Olive et al., 2007) but repress the Salmonella T3SS (Wang et al., 2010); Listeria are resistant (Begley et al., 2003). These factors tend to be incorporated only when they are the subject of investigation.

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Polarized in vitro organic culture (pIVOC) systems represent an advance over traditional IVOC by limiting interactions between bacteria and host cells to the relevant surface. A recent study of EPEC interaction with the human small intestine in a pIVOC system provided (i) enhanced colonization compared with standard IVOC, (ii) evidence of bacterial detachment, as in natural rabbit EPEC infections and (iii) indications that EPEC can trigger mucosal IL-8 responses by apical flagellin/TLR5 interaction ex vivo and does not require access to the basolateral membrane as suggested by cell culture models (Schu¨ller et al., 2009). 3.6. In vivo systems Because in vitro systems lack the complete environment and the full repertoire of host defence mechanisms, in vivo experiments are conducted to gain greater insight into what really happens in disease. Several systems that vary in complexity and in the information they can provide are in use. Biopsy material permits examination of a point in time of a natural or experimental infection, devoid of many of the artificial circumstances that characterize in vitro systems. It is not without its challenges and its own set of problems, including anesthesia, technical expertise, and a limited amount of tissues. Surrogate hosts such as Caenorhabditis elegans are used to identify bacterial virulence factors and innate immune defences against bacterial pathogens. Straightforward infection of intact animals is frequently used but questions of dose, method, route and frequency of administration, and method of preparation of the inoculum may markedly affect the outcome. Experimental infections include the use of models such as ligated intestine and skin pouches. Competition during mixed infection of mutant and wild type parent is a valuable model for evaluating the contribution of specific genes to pathogenesis. Germ-free animals facilitate experimental infection but are costly and provide information which may be different from that obtained when the host has a normal flora. Bacteria may be modified to create an animal model. For example, substitutions in 2 amino acids of L. monocytogenes internalin increased the binding affinity for mouse E-cadherin 5000-fold and allowed the mouse to be used as a model of human listeriosis (Wollert et al., 2007). Natural infections represent the ultimate setting for the disease processes we are trying to understand. However, investigating such infections is fraught with difficulties – variables such as time of infection, dose, and duration of exposure are uncontrolled and there may be practical difficulties in accessing infected animals. Nonetheless, there is enormous value in regularly checking data obtained in the various artificially constructed experimental systems with what is going on in the course of natural disease. We cannot always rely strictly on the literature as a basis for comparison as pathogenesis is a moving target; many infectious diseases of animals change over time. We take note of those in which dramatic change has occurred but those in which small changes have occurred are likely to go unnoticed.

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4. Conclusions Complexity is to be expected in pathogenesis. Consider, for example, that a bacterium with 20 genes for surface proteins that can be expressed in two forms could generate 220 (over 1 million) possible combinations of these proteins capable of interacting with the host. Regulation is a key aspect of bacterial efficiency, operating not only on an on/off basis but also as rheostats that calibrate response. Regulation is a major component of the complexity, directing the activity of pathways that involve vast networks, many intersections, and constantly changing environments. Complexity can lead to findings that are irrelevant in the appropriate host–pathogen–disease context. Collaborations among scientists who bring a range of specific expertise to bear on a pathogenesis problem are necessary to satisfactorily address the complexities of pathogenesis. The best chances for determining relevant information on bacterial pathogenesis exist when there is careful planning to ensure that relevant questions are being asked, that the appropriate test systems are used, and that the limitations of the system and the data are recognized. Use of a combination of techniques, disciplines, and minds facilitates the process. Secondary pathologies, such as cancer and autoimmune diseases, co-infections, and polymicrobic communities, including viruses, are added complications that need to be considered in interpreting the results of pathogenesis research. Conflict of interest statement There was no funding and no conflicts of interest associated with this manuscript. References Agarwal, N., Lamichhane, G., Gupta, R., Nolan, S., Bishai, W.R., 2009. Cyclic AMP intoxication of macrophages by a Mycobacterium tuberculosis adenylate cyclase. Nature 460 (7251), 98–102. Almazan, J., de la Fuente, R., Gomez-Lucia, E., Suarez, G., 1987. Enterotoxin production by strains of Staphylococcus intermedius and Staphylococcus aureus isolated from dog infections. Zentralbl. Bakteriol. Mikrobiol. Hyg. A 264 (1–2), 29–32. Alvarez-Martinez, C.E., Christie, P.J., 2009. Biological diversity of prokaryotic type IV secretion systems. Microbiol. Mol. Biol. Rev. 73 (4), 775– 808. Amador-Noguez, D., Feng, X.J., Fan, J., Roquet, N., Rabitz, H., Rabinowitz, J.D., 2010. Systems-level metabolic flux profiling elucidates a complete, bifurcated tricarboxylic acid cycle in Clostridium acetobutylicum. J. Bacteriol. 192 (17), 4452–4461. Ashida, H., Ogawa, M., Mimuro, H., Sasakawa, C., 2009. Shigella infection of intestinal epithelium and circumvention of the host innate defense system. Curr. Top. Microbiol. Immunol. 337, 231–255. Bainbridge, B.W., Darveau, R.P., 2001. Porphyromonas gingivalis lipopolysaccharide: an unusual pattern recognition receptor ligand for the innate host defense system. Acta Odontol. Scand. 59 (3), 131– 138. Baxt, L.A., Goldberg, M.B., 2010. Anaerobic environment of the intestine primes pathogenic Shigella for infection. Expert Rev. Anti Infect. Ther. 8 (11), 1225–1229. Begley, M., Hill, C., Gahan, C.G., 2003. Identification and disruption of btlA, a locus involved in bile tolerance and general stress resistance in Listeria monocytogenes. FEMS Microbiol. Lett. 218 (1), 31– 38. Beaume, M., Hernandez, D., Francois, P., Schrenzel, J., 2010. New approaches for functional genomic studies in staphylococci. Int. J. Med. Microbiol. 300 (2–3), 88–97.

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