Epigallocatechin gallate inhibits Francisella tularensis growth and suppresses the function of DNA-binding protein HU.

Francisella tularensis is a highly infectious intracellular bacterium causing tularemia disease and is regarded as a potential biological weapon. The development of a vaccine, effective treatment, or prophylactic substances targeted against tularemia is in the forefront of interest and could help to prevent or mitigate possible malevolent acts by bioterrorism utilizing F. tularensis. The viability of F. tularensis, and thus of a tularemia disease outbreak, might potentially be suppressed by simple commonly available natural substances. Epigallocatechin gallate (EGCG) is contained in green tea and its antimicrobial effect has been described. Here, we show that EGCG can suppress F. tularensis growth and is able to reduce the bacterium's ability to replicate inside mouse bone marrow-derived macrophages (BMMs) without side effects on BMMs' own viability. We suggest one (but not the only) mechanism of EGCG action. We demonstrate that EGCG can block the main functions of HU protein, the important regulator of F. tularensis virulence, leading to overall attenuation of F. tularensis viability. EGCG can delay death of mice infected by F. tularensis and can be used as a prophylactic agent against tularemia disease. Postponing death by up to 2 days can provide sufficient opportunity to administer another treatment agent.

Characterization of tetratricopeptide repeat-like proteins in Francisella tularensis and identification of a novel locus required for virulence.

Francisella tularensis is a highly infectious bacterium that causes the potentially lethal disease tularemia. This extremely virulent bacterium is able to replicate in the cytosolic compartments of infected macrophages. To invade macrophages and to cope with their intracellular environment, Francisella requires multiple virulence factors, which are still being identified. Proteins containing tetratricopeptide repeat (TPR)-like domains seem to be promising targets to investigate, since these proteins have been reported to be directly involved in virulence-associated functions of bacterial pathogens. Here, we studied the role of the FTS_0201, FTS_0778, and FTS_1680 genes, which encode putative TPR-like proteins in Francisella tularensis subsp. holarctica FSC200. Mutants defective in protein expression were prepared by TargeTron insertion mutagenesis. We found that the locus FTS_1680 and its ortholog FTT_0166c in the highly virulent Francisella tularensis type A strain SchuS4 are required for proper intracellular replication, full virulence in mice, and heat stress tolerance. Additionally, the FTS_1680-encoded protein was identified as a membrane-associated protein required for full cytopathogenicity in macrophages. Our study thus identifies FTS_1680/FTT_0166c as a new virulence factor in Francisella tularensis.

Francisella tularensis subsp. holarctica DsbA homologue: a thioredoxin-like protein with chaperone function.

Francisella tularensis is a highly infectious facultative intracellular bacterium and aetiological agent of tularaemia. The conserved hypothetical lipoprotein with homology to thiol/disulphide oxidoreductase proteins (FtDsbA) is an essential virulence factor in F. tularensis. Its protein sequence has two different domains: the DsbA_Com1_like domain (DSBA), with the highly conserved catalytically active site CXXC and cis-proline residue; and the domain amino-terminal to FKBP-type peptidyl-prolyl isomerases (FKBP_N). To establish the role of both domains in tularaemia infection models, site-directed and deletion mutagenesis affecting the active site (AXXA), the cis-proline (P286T) and the FKBP_N domain (ΔFKBP_N) were performed. The generated mutations led to high attenuation with the ability to induce full or partial host protective immunity. Recombinant protein analysis revealed that the active site CXXC as well as the cis-proline residue and the FKBP_N domain are necessary for correct thiol/disulphide oxidoreductase activity. By contrast, only the DSBA domain (and not the FKBP_N domain) seems to be responsible for the in vitro chaperone activity of the FtDsbA protein.

Bacteriophage SPO1 protein Gp46 suppresses functions of HU protein in Francisella tularensis.

The nucleoid-associated protein HU is a common bacterial transcription factor, whose role in pathogenesis and virulence has been described in many bacteria. Our recent studies showed that the HU protein is an indispensable virulence factor in the human pathogenic bacterium Francisella tularensis, a causative agent of tularemia disease, and that this protein can be a key target in tularemia treatment or vaccine development. Here, we show that Francisella HU protein is inhibited by Gp46, a protein of Bacillus subtilis bacteriophage SPO1. We predicted that Gp46 could occupy the F. tularensis HU protein DNA binding site, and subsequently confirmed the ability of Gp46 to abolish the DNA-binding capacity of HU protein. Next, we showed that the growth of Francisella wild-type strain expressing Gp46 in trans corresponded to that of a deletion mutant strain lacking the HU protein. Similarly, the efficiency of intracellular proliferation in mouse macrophages resembled that of the deletion mutant strain, but not that of the wild-type strain. These results, in combination with findings from a recent study on Gp46, enabled us to confirm that Gp46 could be a universal inhibitor of HU proteins among bacterial species.

Modified activities of macrophages' deubiquitinating enzymes after Francisella infection.

Francisella tularensis influences several host molecular/signaling pathways during infection. Ubiquitination and deubiquitination are among the most important regulatory mechanisms and respectively occur through attachment or removal of the ubiquitin molecule. The process is necessary not only to mark molecules for degradation, but also, for example, to the activation of signaling pathways leading to pro-inflammatory host response. Many intracellular pathogens, including Francisella tularensis, have evolved mechanisms of modifying such host immune responses to escape degradation. Here, we describe that F. tularensis interferes with the host's ubiquitination system. We show increased total activity of deubiquitinating enzymes (DUBs) in human macrophages after infection, while confirm reduced enzymatic activities of two specific DUBs (USP10 and UCH-L5), and demonstrate increased activity of USP25. We further reveal the enrichment of these three enzymes in exosomes derived from F. tularensis-infected cells. The obtained results show the regulatory effect on ubiquitination mechanism in macrophages during F. tularensis infection.

Arginine 58 is indispensable for proper function of the Francisella tularensis subsp. holarctica FSC200 HU protein, and its substitution alters virulence and mediates immunity against wild-type strain.

HU protein, a member of the nucleoid-associated group of proteins, is an important transcription factor in bacteria, including in the dangerous human pathogen Francisella tularensis. Generally, HU protein acts as a DNA sequence non-specific binding protein and it is responsible for winding of the DNA chain that leads to the separation of transcription units. Here, we identified potential HU protein binding sites using the ChIP-seq method and two possible binding motifs in F. tularensis subsp. holarctica FSC200 depending upon growth conditions. We also confirmed that FSC200 HU protein is able to introduce negative supercoiling of DNA in the presence of topoisomerase I. Next, we showed interaction of the HU protein with a DNA region upstream of the pigR gene and inside the clpB gene, suggesting possible regulation of PigR and ClpB expression. Moreover, we showed that arginine 58 and partially arginine 61 are important for HU protein's DNA binding capacity, negative supercoiling induction by HU, and the length and winding of FSC200 chromosomal DNA. Finally, in order to verify biological function of the HU protein, we demonstrated that mutations in arginine 58, arginine 61, and serine 74 of the HU protein decrease virulence of FSC200 both in vitro and in vivo and that immunization using these mutant strains is able to protect as many as 100% of mice against wild-type challenge. Taken together, our findings deepen knowledge about the role of the HU protein in tularaemia pathogenesis and suggest that HU protein should be addressed in the context of tularaemia vaccine development.

Bacterial nucleoid-associated protein HU as an extracellular player in host-pathogen interaction.

HU protein is a member of nucleoid-associated proteins (NAPs) and is an important regulator of bacterial virulence, pathogenesis and survival. NAPs are mainly DNA structuring proteins that influence several molecular processes by binding the DNA. HU´s indispensable role in DNA-related processes in bacteria was described. HU protein is a necessary bacterial transcription factor and is considered to be a virulence determinant as well. Less is known about its direct role in host-pathogen interactions. The latest studies suggest that HU protein may be secreted outside bacteria and be a part of the extracellular matrix. Moreover, HU protein can be internalized in a host cell after bacterial infection. Its role in the host cell is not well described and further studies are extremely needed. Existing results suggest the involvement of HU protein in host cell immune response modulation in bacterial favor, which can help pathogens resist host defense mechanisms. A better understanding of the HU protein's role in the host cell will help to effective treatment development.

Identification of multiple substrates of the StkP Ser/Thr protein kinase in Streptococcus pneumoniae.

Monitoring the external environment and responding to its changes are essential for the survival of all living organisms. The transmission of extracellular signals in prokaryotes is mediated mainly by two-component systems. In addition, genomic analyses have revealed that many bacteria contain eukaryotic-type Ser/Thr protein kinases. The human pathogen Streptococcus pneumoniae encodes 13 two-component systems and has a single copy of a eukaryotic-like Ser/Thr protein kinase gene designated stkP. Previous studies demonstrated the pleiotropic role of the transmembrane protein kinase StkP in pneumococcal physiology. StkP regulates virulence, competence, and stress resistance and plays a role in the regulation of gene expression. To determine the intracellular signaling pathways controlled by StkP, we used a proteomic approach for identification of its substrates. We detected six proteins phosphorylated on threonine by StkP continuously during growth. We identified three new substrates of StkP: the Mn-dependent inorganic pyrophosphatase PpaC, the hypothetical protein spr0334, and the cell division protein DivIVA. Contrary to the results of a previous study, we did not confirm that the alpha-subunit of RNA polymerase is a target of StkP. We showed that StkP activation and substrate recognition depend on the presence of a peptidoglycan-binding domain comprising four extracellular penicillin-binding protein- and Ser/Thr kinase-associated domain (PASTA domain) repeats. We found that StkP is regulated in a growth-dependent manner and likely senses intracellular peptidoglycan subunits present in the cell division septa. In addition, stkP inactivation results in cell division defects. Thus, the data presented here suggest that StkP plays an important role in the regulation of cell division in pneumococcus.

Control of Francisella tularensis Virulence at Gene Level: Network of Transcription Factors.

Regulation of gene transcription is the initial step in the complex process that controls gene expression within bacteria. Transcriptional control involves the joint effort of RNA polymerases and numerous other regulatory factors. Whether global or local, positive or negative, regulators play an essential role in the bacterial cell. For instance, some regulators specifically modify the transcription of virulence genes, thereby being indispensable to pathogenic bacteria. Here, we provide a comprehensive overview of important transcription factors and DNA-binding proteins described for the virulent bacterium Francisella tularensis, the causative agent of tularemia. This is an unexplored research area, and the poorly described networks of transcription factors merit additional experimental studies to help elucidate the molecular mechanisms of pathogenesis in this bacterium, and how they contribute to disease.

Nucleoid-Associated Protein HU: A Lilliputian in Gene Regulation of Bacterial Virulence.

Nucleoid-associated proteins belong to a group of small but abundant proteins in bacterial cells. These transcription regulators are responsible for many important cellular processes and also are involved in pathogenesis of bacteria. The best-known nucleoid-associated proteins, such as HU, FIS, H-NS, and IHF, are often discussed. The most important findings in research concerning HU protein are described in this mini review. Its roles in DNA compaction, shape modulation, and negative supercoiling induction have been studied intensively. HU protein regulates bacteria survival, growth, SOS response, virulence genes expression, cell division, and many other cell processes. Elucidating the mechanism of HU protein action has been the subject of many research projects. This mini review provides a comprehensive overview of the HU protein.

HU protein is involved in intracellular growth and full virulence of Francisella tularensis.

The nucleoid-associated HU proteins are small abundant DNA-binding proteins in bacterial cell which play an important role in the initiation of DNA replication, cell division, SOS response, control of gene expression and recombination. HU proteins bind to double stranded DNA non-specifically, but they exhibit high affinity to abnormal DNA structures as four-way junctions, gaps or nicks, which are generated during DNA damage. In many pathogens HU proteins regulate expression of genes involved in metabolism and virulence. Here, we show that the Francisella tularensis subsp. holarctica gene locus FTS_0886 codes for functional HU protein which is essential for full Francisella virulence and its resistance to oxidative stress. Further, our results demonstrate that the recombinant FtHU protein binds to double stranded DNA and protects it against free hydroxyl radicals generated via Fenton's reaction. Eventually, using an iTRAQ approach we identified proteins levels of which are affected by the deletion of hupB, among them for example Francisella pathogenicity island (FPI) proteins. The pleiotropic role of HU protein classifies it as a potential target for the development of therapeutics against tularemia.

Francisella tularensis D-Ala D-Ala Carboxypeptidase DacD Is Involved in Intracellular Replication and It Is Necessary for Bacterial Cell Wall Integrity.

D-alanyl-D-alanine carboxypeptidase, product of dacD gene in Francisella, belongs to penicillin binding proteins (PBPs) and is involved in remodeling of newly synthetized peptidoglycan. In E. coli, PBPs are synthetized in various growth phases and they are able to substitute each other to a certain extent. The DacD protein was found to be accumulated in fraction enriched in membrane proteins from severely attenuated dsbA deletion mutant strain. It has been presumed that the DsbA is not a virulence factor by itself but that its substrates, whose correct folding and topology are dependent on the DsbA oxidoreductase and/or isomerase activities, are the primary virulence factors. Here we demonstrate that Francisella DacD is required for intracellular replication and virulence in mice. The dacD insertion mutant strain showed higher sensitivity to acidic pH, high temperature and high osmolarity when compared to the wild-type. Eventually, transmission electron microscopy revealed differences in mutant bacteria in both the size and defects in outer membrane underlying its SDS and serum sensitivity. Taken together these results suggest DacD plays an important role in Francisella pathogenicity.

Identification of two substrates of FTS_1067 protein - An essential virulence factor of Francisella tularensis.

Francisella tularensis is a highly virulent intracellular pathogen with the capacity to infect a variety of hosts including humans. One of the most important proteins involved in F. tularensis virulence and pathogenesis is the protein DsbA. This protein is annotated as a lipoprotein with disulfide oxidoreductase/isomerase activity. Therefore, its interactions with different substrates, including probable virulence factors, to assist in their proper folding are anticipated. We aimed to use the immunopurification approach to find DsbA (gene locus FTS_1067) interacting partners in F. tularensis subsp. holarctica strain FSC200 and compare the identified substrates with proteins which were found in our previous comparative proteome analysis. As a result of our work two FTS_1067 substrates, D-alanyl-D-alanine carboxypeptidase family protein and HlyD family secretion protein, were identified. Bacterial two-hybrid systems were further used to test their relevance in confirming FTS_1067 protein interactions.

Cooperation of both, the FKBP_N-like and the DSBA-like, domains is necessary for the correct function of FTS_1067 protein involved in Francisella tularensis virulence and pathogenesis.

Francisella tularensis the etiological agent of tularaemia is one of the most infectious human pathogen known. Our knowledge about its key virulence factors has increased recently but it still remains a lot to explore. One of the described essential virulence factors is membrane lipoprotein FTS_1067 (nomenclature of F. tularensis subsp. holarctica strain FSC200) with homology to the protein family of disulphide oxidoreductases DsbA. Lipoprotein consists of two different domains: the C-terminal DsbA_Com1-like domain (DSBA-like) and the N-terminal FKBP-type peptidyl-prolyl cis/trans isomerases (FKBP_N-like). To uncover the biological role of these domains, we created bacterial strain with deletion of the DSBA-like domain. This defect in gene coding for lipoprotein FTS_1067 led to high in vivo attenuation associated with the ability to induce host protective immunity. Analyses performed with the truncated recombinant protein showed that the absence of DSBA-like domain revealed the loss of thiol/disulphide oxidoreductase activity and, additionally, confirmed the role of the FKBP_N-like domain in the FTS_1067 oligomerization and chaperone-like function. Finally, we verified that only full-length form of FTS_1067 recombinant protein possesses the isomerase activity. Based on our results, we proposed that for the correct FTS_1067 protein function both domains are needed.

Francisella tularensis type B ΔdsbA mutant protects against type A strain and induces strong inflammatory cytokine and Th1-like antibody response in vivo.

Francisella tularensis subspecies tularensis is a highly virulent intracellular bacterial pathogen, causing the disease tularemia. However, a safe and effective vaccine for routine application against F. tularensis has not yet been developed. We have recently constructed the deletion mutants for the DsbA homolog protein (ΔdsbA/FSC200) and a hypothetical protein IglH (ΔiglH/FSC200) in the type B F. tularensis subsp. holarctica FSC200 strain, which exerted different protection capacity against parental virulent strain. In this study, we further investigated the immunological correlates for these different levels of protection provided by ΔdsbA/FSC200 and ΔiglH/FSC200 mutants. Our results show that ΔdsbA/FSC200 mutant, but not ΔiglH/FSC200 mutant, induces an early innate inflammatory response leading to strong Th1-like antibody response. Furthermore, vaccination with ΔdsbA/FSC200 mutant, but not with ΔiglH/FSC200, elicited protection against the subsequent challenge with type A SCHU S4 strain in mice. An immunoproteomic approach was used to map a spectrum of antigens targeted by Th1-like specific antibodies, and more than 80 bacterial antigens, including novel ones, were identified. Comparison of tularemic antigens recognized by the ΔdsbA/FSC200 post-vaccination and the SCHU S4 post-challenge sera then revealed the existence of 22 novel SCHU S4 specific antibody clones.

Deletion of IglH in virulent Francisella tularensis subsp. holarctica FSC200 strain results in attenuation and provides protection against the challenge with the parental strain.

Francisella tularensis, the causative agent of tularemia, is a highly infectious intracellular pathogen with no licensed vaccine available today. The recent search for genome sequences involved in F. tularensis virulence mechanisms led to the identification of the 30-kb region defined as a Francisella pathogenicity island (FPI). In our previous iTRAQ study we described the concerted upregulation of some FPI proteins in different F. tularensis strains cultivated under stress conditions. Among them we identified the IglH protein whose role in Francisella virulence has not been characterized yet. In this work we deleted the iglH gene in a European clinical isolate of F. tularensis subsp. holarctica FSC200. We showed that the iglH gene is necessary for intracellular growth and escape of F. tularensis from phagosomes. We also showed that the iglH mutant is avirulent in a mouse model of infection and persists in the organs for about three weeks after infection. Importantly, mice vaccinated by infection with the iglH mutant were protected against subcutaneous challenge with the fully virulent parental FSC200 strain. This is the first report of a defined subsp. holarctica FPI deletion strain that provides protective immunity against subsequent subcutaneous challenge with a virulent isolate of F. tularensis subsp. holarctica.