Use of in vivo-induced antigen technology (IVIAT) for the identification of Streptococcus suis serotype 2 in vivo-induced bacterial protein antigens

Streptococcus suis (S. suis) infections have been considered a major problem in the swine industry worldwide, particularly over the past 20 years. S. suis is a gram-positive, facultatively anaerobic coccus, and 35 serotypes (1-34 and 1/2) have been described based on their capsular antigens. Among these, serotype 2 (SS2) is the causative agent of many different syndromes worldwide, including meningitis, septicemia, arthritis, and pneumonia in humans, swine, and other animals [1]. In addition, SS2 is widely recognized as an important zoonotic agent that afflicts people in close contact with infected pigs or pork-derived products [2, 3]. Two recent large-scale outbreaks of human streptococcal toxic shock syndrome (STSS) caused by SS2 in China in 1998 and in 2005 have increased public health concerns worldwide. Notably, a major outbreak of SS2 infection emerged in the summer of 2005 in Sichuan Province, China. A total of 215 cases of human S. suis infection were reported, and the outbreak resulted in 38 deaths and massive economic losses [4, 5].

Little is known about the virulence factors of SS2. To date, only a few SS2 virulence associated factors have been identified and characterized; these include the capsular polysaccharide (CPS) [1], suilysin (SLY) [6], muramidase-released protein (MRP) [7], extracellular protein factor (EF) [8], adhesin [9], cell wall-associated and extracellular proteins [10], fibronectin- and fibrinogen-binding protein (FBP) [11], a serum opacity factor [12], and the arginine deiminase system [13, 14].

An understanding of SS2-host molecular interactions is crucial for understanding SS2 pathogenesis and immunology. Conventional genetic and biochemical approaches used to study SS2 virulence factors are unable to take into account in the complex and dynamic environmental stimuli associated with the infection process. Recently, several technologies, including in vivo expression technology (IVET), differential fluorescence induction (DFI), signature-tagged mutagenesis (STM), transcriptional and proteomic profiling, and in vivo-induced antigen technology (IVIAT) have been developed to identify the pathogen genes expressed during the infection process [15, 16].

IVIAT is a method that allows for the direct identification of microbial proteins expressed at sufficient levels during host infection to be immunogenic. A schematic of the IVIAT procedure was described by Rollins et al [16]. The advantage of IVIAT is that it enables the identification of antigens expressed specifically during infection, but not during growth in standard laboratory media. It was speculated that the genes and gene pathways identified by IVIAT may play a role in virulence or pathogenesis during bacterial infection [17, 18]. IVIAT has been successfully used to identify arrays of in vivo induced proteins in Salmonella enterica serovar Typhi [19], Escherichia coli O157 [18], Group A Streptococcus (GAS) [17], Vibrio cholerae [20], and others, and these proteins have been shown to contribute to the pathogenesis or virulence of the infecting organisms. When IVIAT was applied to E. coli O157, it identified 223 O157 proteins expressed during human infection. Among these, four proteins--intimin-γ (an adhesin), QseA (a quorum-sensing transcriptional regulator), TagA (a lipoprotein), and MsbB2 (an acyltransferase)--had been previously identified as virulence-related proteins [18].

To identify SS2 proteins that are immunogenic and expressed uniquely during SS2 infection, we applied the newly developed and modified IVIAT method. Briefly, we screened a library of SS2 proteins expressed in E. coli to identify clones that were immunoreactive with convalescent-phase sera, which had been previously fully adsorbed against in vitro-grown SS2 and E. coli organisms. The adsorption process eliminates antibodies reactive with in vitro-expressed antigens and allows for the identification of clones expressing protein antigens that are upregulated during swine infection. Specifically, we hypothesized that by using IVIAT, we could identify proteins that play a role in the SS2-specific host-bacterium interactions unique to SS2 infection in pigs. In this study, we identified 48 putative in vivo-induced (IVI) proteins, which included proteins associated with bacterial cell wall structure, metabolism, regulation, molecule synthesis, substance transport and others. Of these, 10 genes were selected for analysis by real-time PCR to confirm their in vivo upregulation. Six genes were shown to be upregulated in vivo. These results suggest that these newly identified genes may contribute to SS2 pathogenesis.

S. suis infection is a major cause of sudden death of pigs, and is also increasingly becoming a human health concern due to its zoonotic transmission capabilities. Attempts to control the infection have been hampered by our lack of knowledge about SS2 pathogenicity. The identification and characterization of putative virulence factors and other infection-related proteins will aid in the prevention and control of SS2 disease. IVIAT provides a "snapshot" of protein expression during infection, allowing us a glimpse into the possible mechanisms by which this pathogen might counter host defenses and adapt and establish itself within the host to cause disease [18]. In the present study, we used the newly developed IVIAT method to select in vivo-induced proteins.

Convalescent-phase sera collected from pigs naturally infected with SS2 are ideal for IVIAT [16]. However, for multiple reasons, it is not easy to acquire convalescent-phase sera from SS2 natural infection. First, epidemiological studies have found that SS2 outbreaks are usually infrequent and only affect a small number of pigs, which can lead to underdiagnosis or misdiagnosis. Second, pigs infected with SS2 do not always show obvious clinical symptoms, and may become carriers without showing clinical signs. Finally, based on its polysaccharide capsular antigens, at least 35 serotypes of S. suis exist. Isolates belonging to other serotypes (such as 1, 1/2, 3, 4, 5, 7, 8 and 9) have also been associated with disease in pigs [28, 29]. Common antigens had been found to be shared between SS2 and these other serotypes (unpublished data from our lab). To reduce these possible interferences, we used pigs with clear backgrounds as animal models, and convalescent sera were prepared following artificial infection.

Until recently, the exact mechanism of SS2 transmission (from pig to human or between pigs) was still poorly understood, but was thought to involve aerosol transmission or other pathways [28–30]. However, some hypotheses about the critical stages of the infection, such as bacterial invasion from the mucosal surfaces to the bloodstream, survival of the bacteria in blood, and invasion from blood into the central nervous system have been presented [28]. Regardless of the mechanism of SS2 invasion, circulation in the blood plays an important role during SS2 disease development. In addition, S. suis is an agent of zoonosis, afflicting people in close contact with infected pigs or pork-derived products. The organisms probably gain entry via small wounds or through inhalation [4, 10, 29]. Furthermore, transmission between pigs in herds through cutaneous wounds has been suggested [29]. In light of these considerations, intravenous and intramuscular inoculations were employed to assay the expression of SS2 in vivo, and to try to mimic natural infection (such as the middle or late stage of the infection).

In this study, we used real-time PCR to analyze the induction of the expression of IVI genes under different environmental conditions. Real-time PCR results demonstrated that the expression of six of the 10 selected genes was upregulated under in vivo conditions. The upregulation time points for these six genes were 12, 24, and 36 h for ss-1616 and trag, 24 h for hprk and sdh, and 36 h for nlpa and ss-1298. This upregulated expression suggests that these genes may play a significant role during the course of SS2 infection (middle, late, or whole stage of infection). The expression profiles of the other four genes (ysirk, srt, cwh, and ss-1955) showed that they were not obviously upregulated under the in vivo condition (Figure 3). There are two possible explanations for this result. First, since we measured the in vivo gene expression at 12, 24, and 36 h pi, it is possible that we missed the time when the levels of expression of these genes were high relative to the expression of the same gene in vitro. For instance, they may play roles in the rapid replication of SS2 during an early stage of infection. Second, the mRNA concentration might not reflect the amount of protein or antigen produced if these antigens are regulated post-transcriptionally.

The protein TRAG, which is a component of a type IV secretion system (T4SS, virulence associated pathway of SS2), was identified. The T4SS mediates horizontal gene transfer, thus contributing to genome plasticity, the evolution of infectious pathogens, and the dissemination of antibiotic resistance and other virulence traits [31]. The gene trag was found in 98HAH33, 05HAH33, Canada strain 89/1591, and all the tested Chinese SS2 virulent strains, but not in European strain P1/7 or the non-virulent strain T15 (data not shown). Brucella species require a T4SS to reach their proper niche and to replicate within host cells [32]. Whether DNA transfer through a T4SS occurs between SS2 isolates and results in an increase in virulence is unknown, and will only be answered by future studies.

Lipoproteins that are upregulated in vivo in other pathogenic organisms have been identified, and have been shown to be likely important for pathogenesis [33, 34]. For instance, in a previous study of Vibrio vulnificus using IVIAT, a putative lipoprotein was also found to be induced in vivo when convalescent-phase sera from patients who survived V. vulnificus septicemia were used to screen a genomic library of this organism [35]. As for nlpa, almost nothing is known about the homolog of this gene in the NCBI database. The partial NLPA protein sequence was identified as a lipoprotein in the 89/1591 genome database, and shares 100% identity with a putative NLPA.

HPr kinase/P-Ser-HPr phosphatase (HprK/P) is a phosphocarrier protein of the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS). It is also a sensor of two-component signal transduction systems (TCSs) [36]. Thus, HprK/P provides a link between carbon metabolism and the development of virulence [37]. For example, the expression of several virulence genes, such as the hemolysin-encoding hly and the phospholipase-encoding plcA, is repressed when Listeria monocytogenes is grown on cellobiose, glucose, fructose, or other rapidly metabolizable carbon sources [38].

L-Serine dehydratase, an iron-sulfur protein [39], was identified, and this gene was also found in the Canadian strain 89/1591. During the course of the infection, alternative carbon sources (such as amino acids) are utilized by bacteria for growth due to competition for nutrients. The results of Velayudhan et al. showed that the catabolism of L-serine by serine dehydratase was crucial for the growth of Campylobacter jejuni under in vivo conditions [40]. In addition, the fermentation of amino acids produces ammonia that neutralizes the surrounding pH; this neutralization is beneficial since it protects group A Streptococcus (GAS) from acid-induced damage [41].

The protein encoded by ss-1298 is an ABC-type transporter, but its function is still unknown. Gene ss-1616 is a conserved hypothetical outer membrane protein in SS2 genome database, and almost nothing is known about this gene. It was found in all tested strains in this study, and in Canada strain 89/1591 and European strain P1/7.

Many surface proteins of pathogenic gram-positive bacteria are linked to the cell wall envelope by a sorting mechanism that recognizes an LPXTG motif, but surface proteins of Streptococcus pneumoniae harbor another motif, YSIRK-G/S [42]. About 20 surface proteins of Staphylococcus aureus carry the YSIRK-G/S motif, whereas those of Listeria monocytogenes and Bacillus anthracis do not [43, 44]. While the function of the YSIRK motif has not been completely elucidated, it may contribute to the efficient secretion of a protein [43]. In the present study, four clones encoded two proteins containing this motif. Although the gene ysirk was only detected 12 h after SS2 infection and then disappeared, and was not strongly upregulated in vivo, the mature protein encoded by ysirk1 showed homology to the surface-associated subtilisin-like serine protease PrtA (a virulence factor) of S. pneumoniae[21]. However, the role of this protein during SS2 infection remains to be determined.

IVIAT enables the identification both of proteins expressed specifically during host infection but not during growth under standard laboratory conditions, and of proteins expressed at significantly higher levels in vivo than in vitro. But IVIAT has its own limitations. IVIAT will not identify all virulence-associated genes. Genes that are expressed both in vivo and in vitro and genes that are not expressed effectively in the E. coli host expression system will not be identified. For instance, some previously reported SS2 virulence factors, such as MRP, EF, FBPs, CPS, and SLY, could not be screened out by IVIAT in this study. We speculate that they are expressed in both in vivo and in vitro growth conditions, and therefore antibodies specific to these antigens had been eliminated during the convalescent sera adsorption steps.

Unexpectedly, some of the genes identified are likely expressed during in vitro growth conditions, such as DNA polymerase I and III, Primosomal protein n, protein Cpn60, and SMC protein (essential for bacterial cell division and cell wall biosynthesis). We speculate that perhaps their expression level was higher during in vivo growth than in vitro growth, and therefore they were detected by the IVIAT.

Taken together, our results suggest that during the course of infection, bacterial metabolism, envelope composition, and virulence will be adjusted for bacteria to survive in the hostile environment. Bacterial pathogens sense their environment, and in response, genes are induced or repressed through spatial and temporal regulation. Genes and gene pathways identified by IVIAT may play a role in virulence or pathogenesis during host infection, and may be appropriate for inclusion in therapeutic, vaccine, or diagnostic applications. The results of this study yielded a set of potentially valuable proteins of a manageable number for future studies on SS2 pathogenicity and for the development of specific diagnostics and vaccines.