Crystal structures of YeiE from Cronobacter sakazakii and the role of sulfite tolerance in gram-negative bacteria.

SignificanceYeiE has been identified as a master virulence factor of Cronobacter sakazakii. In this study, we determined the crystal structures of the regulatory domain of YeiE in complex with its physiological ligand sulfite ion (SO32-). The structure provides the basis for the molecular mechanisms for sulfite sensing and the ligand-dependent conformational changes of the regulatory domain. The genes under the control of YeiE in response to sulfite were investigated to reveal the functional roles of YeiE in the sulfite tolerance of the bacteria. We propose the molecular mechanism underlying the ability of gram-negative pathogens to defend against the innate immune response involving sulfite, thus providing a strategy to control the pathogenesis of bacteria.

Isocitrate binds to the itaconic acid-responsive LysR-type transcriptional regulator RipR in Salmonella pathogenesis.

Macrophages produce itaconic acid in phagosomes in response to bacterial cell wall component lipopolysaccharide to eliminate invading pathogenic bacteria. Itaconic acid competitively inhibits the first enzyme of the bacterial glyoxylate cycle. To overcome itaconic acid stress, bacteria employ the bacterial LysR-type transcriptional regulator RipR. However, it remains unknown which molecule activates RipR in bacterial pathogenesis. In this study, we determined the crystal structure of the regulatory domain of RipR from the intracellular pathogen Salmonella. The RipR regulatory domain structure exhibited the typical dimeric arrangement with the putative ligand-binding site between the two subdomains. Our isothermal titration calorimetry experiments identified isocitrate as the physiological ligand of RipR, whose intracellular level is increased in response to itaconic acid stress. We further found that 3-phenylpropionic acid significantly decreased the resistance of the bacteria to an itaconic acid challenge. Consistently, the complex structure revealed that the compound is antagonistically bound to the RipR ligand-binding site. This study provides the molecular basis of bacterial survival in itaconic acid stress from our immune systems. Further studies are required to reveal biochemical activity, which would elucidate how Salmonella survives in macrophage phagosomes by defending against itaconic acid inhibition of bacterial metabolism.

Bacteriophage and endolysin engineering for biocontrol of food pathogens/pathogens in the food: recent advances and future trends.

Despite advances in modern technologies, various foodborne outbreaks have continuously threatened the food safety. The overuse of and abuse/misuse of antibiotics have escalated this threat due to the prevalence of multidrug-resistant (MDR) pathogens. Therefore, the development of new methodologies for controlling microbial contamination is extremely important to ensure the food safety. As an alternative to antibiotics, bacteriophages(phages) and derived endolysins have been proposed as novel, effective, and safe antimicrobial agents and applied for the prevention and/or eradication of bacterial contaminants even in foods and food processing facilities. In this review, we describe recent genetic and protein engineering tools for phages and endolysins. The major aim of engineering is to overcome limitations such as a narrow host range, low antimicrobial activity, and low stability of phages and endolysins. Phage engineering also aims to deter the emergence of phage resistance. In the case of endolysin engineering, enhanced antibacterial ability against Gram-negative and Gram-positive bacteria is another important goal. Here, we summarize the successful studies of phages and endolysins treatment in different types of food. Moreover, this review highlights the recent advances in engineering techniques for phages and endolysins, discusses existing challenges, and suggests technical opportunities for further development, especially in terms of antimicrobial agents in the food industry.

From microbes to molecules: a review of microbial-driven antioxidant peptide generation.

Oxidative stress, arising from excess reactive oxygen species (ROS) or insufficient antioxidant defenses, can damage cellular components, such as lipids, proteins, and nucleic acids, resulting in cellular dysfunction. The relationship between oxidative stress and various health disorders has prompted investigations into potent antioxidants that counteract ROS's detrimental impacts. In this context, antioxidant peptides, composed of two to twenty amino acids, have emerged as a unique group of antioxidants and have found applications in food, nutraceuticals, and pharmaceuticals. Antioxidant peptides are sourced from natural ingredients, mainly proteins derived from foods like milk, eggs, meat, fish, and plants. These peptides can be freed from their precursor proteins through enzymatic hydrolysis, fermentation, or gastrointestinal digestion. Previously published studies focused on the origin and production methods of antioxidant peptides, describing their structure-activity relationship and the mechanisms of food-derived antioxidant peptides. Yet, the role of microorganisms hasn't been sufficiently explored, even though the production of antioxidant peptides frequently employs a variety of microorganisms, such as bacteria, fungi, and yeasts, which are recognized for producing specific proteases. This review aims to provide a comprehensive overview of microorganisms and their proteases participating in enzymatic hydrolysis and microbial fermentation to produce antioxidant peptides. This review also covers endogenous peptides originating from microorganisms. The information obtained from this review might guide the discovery of novel organisms adept at generating antioxidant peptides.

Development of a Bacteriophage Cocktail against Pectobacterium carotovorum Subsp. carotovorum and Its Effects on Pectobacterium Virulence.

Pectobacterium carotovorum subsp. carotovorum is a necrotrophic plant pathogen that secretes plant cell wall-degrading enzymes (PCWDEs) that cause soft rot disease in various crops. Bacteriophages have been under consideration as harmless antibacterial agents to replace antibiotics and copper-based pesticides. However, the emergence of bacteriophage resistance is one of the main concerns that should be resolved for practical phage applications. In this study, we developed a phage cocktail with three lytic phages that recognize colanic acid (phage POP12) or flagella (phages POP15 and POP17) as phage receptors to minimize phage resistance. The phage cocktail effectively suppressed the emergence of phage-resistant P. carotovorum subsp. carotovorum compared with single phages in in vitro challenge assays. The application of the phage cocktail to napa cabbage (Brassica rapa subsp. pekinensis) resulted in significant growth retardation of P. carotovorum subsp. carotovorum (P < 0.05) and prevented the symptoms of soft rot disease. Furthermore, phage cocktail treatments of young napa cabbage leaves in a greenhouse environment indicated effective prevention of soft rot disease compared to that in the nonphage negative control. We isolated 15 phage-resistant mutants after a phage cocktail treatment to assess the virulence-associated phenotypes compared to those of wild-type (WT) strain Pcc27. All mutants showed reduced production of four different PCWDEs, leading to lower levels of tissue softening. Ten of the 15 phage-resistant mutants additionally exhibited decreased swimming motility. Taken together, these results show that the phage cocktail developed here, which targets two different types of phage receptors, provides an effective strategy for controlling P. carotovorum subsp. carotovorum in agricultural products, with a potential ability to attenuate P. carotovorum subsp. carotovorum virulence. IMPORTANCE Pectobacterium carotovorum subsp. carotovorum is a phytopathogen that causes soft rot disease in various crops by producing plant cell wall-degrading enzymes (PCWDEs). Although antibiotics and copper-based pesticides have been extensively applied to inhibit P. carotovorum subsp. carotovorum, the emergence of antibiotic-resistant bacteria and demand for harmless antimicrobial products have emphasized the necessity of finding alternative therapeutic strategies. To address this problem, we developed a phage cocktail consisting of three P. carotovorum subsp. carotovorum-specific phages that recognize colanic acids and flagella of P. carotovorum subsp. carotovorum. The phage cocktail treatments significantly decreased P. carotovorum subsp. carotovorum populations, as well as soft rot symptoms in napa cabbage. Simultaneously, they resulted in virulence attenuation in phage-resistant P. carotovorum subsp. carotovorum, which was represented by decreased PCWDE production and decreased flagellum-mediated swimming motility. These results suggested that preparations of phage cocktails targeting multiple receptors would be an effective approach to biocontrol of P. carotovorum subsp. carotovorum in crops.

Structural basis for HOCl recognition and regulation mechanisms of HypT, a hypochlorite-specific transcriptional regulator.

Hypochlorous acid (HOCl) is generated in the immune system to kill microorganisms. In Escherichia coli, a hypochlorite-specific transcription regulator, HypT, has been characterized. HypT belongs to the LysR-type transcriptional regulator (LTTR) family that contains a DNA-binding domain (DBD) and a regulatory domain (RD). Here, we identified a hypT gene from Salmonella enterica serovar Typhimurium and determined crystal structures of the full-length HypT protein and the RD. The full-length structure reveals a type of tetrameric assembly in the LTTR family. Based on HOCl-bound and oxidation-mimicking structures, we identified a HOCl-driven methionine oxidation mechanism, in which the bound HOCl oxidizes a conserved methionine residue lining the putative ligand-binding site in the RD. Furthermore, we proposed a molecular model for the oxidized HypT, where methionine oxidation by HOCl results in a conformational change of the RD, inducing a counter rotation of the DBD dimers. Target genes that are regulated by HypT and their roles in Salmonella were also investigated. DNase I footprinting experiments revealed a DNA segment containing two pseudopalindromic motifs that are separated by ∼100 bp, suggesting that only the oxidized structure makes a concomitant binding, forming a DNA loop. An understanding of the HypT-mediated mechanism would be helpful for controlling many pathogenic bacteria by counteracting bacterial HOCl defense mechanisms.

Structure and function of the hypochlorous acid-induced flavoprotein RclA from Escherichia coli.

In response to microbial invasion, the animal immune system generates hypochlorous acid (HOCl) that kills microorganisms in the oxidative burst. HOCl toxicity is amplified in the phagosome through import of the copper cation (Cu2+). In Escherichia coli and Salmonella, the transcriptional regulator RclR senses HOCl stress and induces expression of the RclA, -B, and -C proteins involved in bacterial defenses against oxidative stress. However, the structures and biochemical roles of the Rcl proteins remain to be elucidated. In this study, we first examined the role of the flavoprotein disulfide reductase (FDR) RclA in the survival of Salmonella in macrophage phagosomes, finding that RclA promotes Salmonella survival in macrophage vacuoles containing sublethal HOCl levels. To clarify the molecular mechanism, we determined the crystal structure of RclA from E. coli at 2.9 Å resolution. This analysis revealed that the structure of homodimeric RclA is similar to those of typical FDRs, exhibiting two conserved cysteine residues near the flavin ring of the cofactor flavin adenine dinucleotide (FAD). Of note, we observed that Cu2+ accelerated RclA-mediated oxidation of NADH, leading to a lowering of oxygen levels in vitro Compared with the RclA WT enzyme, substitution of the conserved cysteine residues lowered the specificity to Cu2+ or substantially increased the production of superoxide anion in the absence of Cu2+ We conclude that RclA-mediated lowering of oxygen levels could contribute to the inhibition of oxidative bursts in phagosomes. Our study sheds light on the molecular basis for how bacteria can survive HOCl stress in macrophages.

An Antibacterial Nanorobotic Approach for the Specific Targeting and Removal of Multiple Drug-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) causes diseases ranging from skin infections to lethal sepsis and has become a serious threat to human health due to multiple-drug resistance (MDR). Therefore, a resistance-free antibacterial therapy is necessary to overcome MDR MRSA infections. In this study, an antibacterial nanorobot (Ab-nanobot) is developed wherein a cell wall-binding domain (CBD)-endolysin, acting as a sensor, is covalently conjugated with an actuator consisting of an iron oxide/silica core-shell. The CBD-endolysin sensor shows an excellent specificity to detect, bind, and accumulate on the S. aureus USA300 cell surface even in a bacterial consortium, and in host cell infections. Ab-nanobot specifically captures and kills MRSA in response to medically approved radiofrequency (RF) electromagnetic stimulation (EMS) signal. When Ab-nanobot receives the RF-EMS signal on the cell surface, actuator induces cell death in MRSA with 99.999% removal within 20 min by cell-wall damage via generation of localized heat and reactive oxygen species. The in vivo efficacy of Ab-nanobot is proven using a mice subcutaneous skin infection model. Collectively, this study offers a nanomedical resistance-free strategy to overcome MDR MRSA infections by providing a highly specific nanorobot for S. aureus.

Regulation of Iron Uptake by Fine-Tuning the Iron Responsiveness of the Iron Sensor Fur.

Iron is one of most abundant environmental metal ions but is highly limited in organisms. It is an important metal ion as it facilitates various biological processes, including catalysis of metabolic enzymes and DNA biogenesis. In bacteria, the ferric uptake regulator (Fur) protein controls iron uptake by regulating genes coding for iron transporters in response to iron concentration. This iron response is ascribed to Fur's intrinsic affinity for iron because its binding to iron dictates its regulatory function. However, we now report that the pathogen Salmonella achieves a proper response of Fur to changes in environmental iron concentrations via EIIANtr (a nitrogen metabolic phosphotransferase system component). We establish that EIIANtr increases expression of iron transporter-coding genes under low-iron conditions (i.e., nanomolar ranges) in a Fur-dependent manner, which promotes Salmonella growth under such conditions. EIIANtr directly hampers Fur binding to DNA, thereby inducing expression of those genes. This regulation allows Salmonella to express Fur-regulated genes under low-iron conditions. Our findings reveal a potentially widespread control mechanism of bacterial iron uptake systems operating in response to iron availability.IMPORTANCE Iron is a fundamental metal ion for living organisms as it facilitates various biological processes. The ferric uptake regulator (Fur) protein controls iron homeostasis in various bacterial species. It is believed that Fur's iron-dependent regulatory action is sufficient for it to function as an iron sensor. However, we now establish that the bacterial pathogen Salmonella enables Fur to properly reflect changes in surrounding iron availability by fine-tuning its responsiveness to iron. This process requires a protein that hampers Fur DNA binding at low iron concentrations. In this way, Salmonella broadens the range of iron concentrations that Fur responds to. Our findings reveal a potentially widespread control mechanism of bacterial iron homeostasis.

LysPBC2, a Novel Endolysin Harboring a Bacillus cereus Spore Binding Domain.

To control the spore-forming human pathogen Bacillus cereus, we isolated and characterized a novel endolysin, LysPBC2, from a newly isolated B. cereus phage, PBC2. Compared to the narrow host range of phage PBC2, LysPBC2 showed very broad lytic activity against all Bacillus, Listeria, and Clostridium species tested. In addition to a catalytic domain and a cell wall binding domain, LysPBC2 has a spore binding domain (SBD) partially overlapping its catalytic domain, which specifically binds to B. cereus spores but not to vegetative cells of B. cereus Both immunogold electron microscopy and a binding assay indicated that the SBD binds the external region of the spore cortex layer. Several amino acid residues required for catalytic or spore binding activity of LysPBC2 were determined by mutagenesis studies. Interestingly, LysPBC2 derivatives with impaired spore binding activity showed an increased lytic activity against vegetative cells of B. cereus compared with that of wild-type LysPBC2. Further biochemical studies revealed that these LysPBC2 derivatives have lower thermal stability, suggesting a stabilizing role of SBD in LysPBC2 structure.IMPORTANCE Bacteriophages produce highly evolved lytic enzymes, called endolysins, to lyse peptidoglycan and release their progeny from bacterial cells. Due to their potent lytic activity and specificity, the use of endolysins has gained increasing attention as a natural alternative to antibiotics. Since most endolysins from Gram-positive-bacterium-infecting phages have a modular structure, understanding the function of each domain is crucial to make effective endolysin-based therapeutics. Here, we report the functional and biochemical characterization of a Bacillus cereus phage endolysin, LysPBC2, which has an unusual spore binding domain and a cell wall binding domain. A single point mutation in the spore binding domain greatly enhanced the lytic activity of endolysin at the cost of reduced thermostability. This work contributes to the understanding of the role of each domain in LysPBC2 and will provide insight for the rational design of efficient antimicrobials or diagnostic tools for controlling B. cereus.

Effective removal of staphylococcal biofilms on various food contact surfaces by Staphylococcus aureus phage endolysin LysCSA13.

Staphylococcal biofilms are recognized as a significant problem in the food industry because of their high resistance to antibiotics, and the use of bacteriophages or endolysins has been regarded as a promising alternative to antibiotics. In this study, LysCSA13, an endolysin from S. aureus virulent bacteriophage CSA13, was cloned and characterized. LysCSA13 consists of an enzymatic active domain and a cell wall binding domain. LysCSA13 showed strong antimicrobial activity against staphylococcal strains at pH 7.0-9.0, 4.0-37.0 °C and in the presence of Ca2+ and Mn2+. In addition, a high efficacy of LysCSA13 in removing staphylococcal biofilms was observed on various surfaces, including polystyrene, glass and stainless steel, displaying an approximately 80-90% decrease in biofilm mass. Furthermore, 300 nM of LysCSA13 effectively removed staphylococcal sessile cells formed on stainless steel and glass by 1-3 log units compared with the untreated control. Scanning electron microscopy analysis visualized the effective deformation and removal of cells embedded in the biofilm matrix. The results indicate that LysCSA13 can effectively control staphylococcal planktonic cells and biofilms regardless of the contact surface matrix and suggest the possible use of LysCSA13 as a promising biocontrol agent in various food processing environments.

Effective inhibition of Salmonella Typhimurium in fresh produce by a phage cocktail targeting multiple host receptors.

Salmonella contamination of fresh produce is the primary bacterial cause of a significant number of foodborne outbreaks and infections. Bacteriophages can be used as natural antibacterial agents to control foodborne pathogens. However, the rapid development of bacterial resistance to phage infection is a significant barrier to practical phage application. To overcome this problem, we developed a novel phage cocktail consisting of the three phages (BSPM4, BSP101 and BSP22A) that target different host receptors, including flagella, O-antigen and BtuB, respectively. Whole genome sequence analysis of the phages revealed that three phages do not harbor genes involved in lysogen formation or toxin production, suggesting they are safe for use as biocontrol agents in foods. In vitro treatment of the phage cocktail resulted in a significant reduction in the development of bacterial resistance. Phage cocktail treatments achieved 4.7-5.5 log CFU/cm2 reduction of viable cell number in iceberg lettuce and 4.8-5.8 log CFU/cm2 reduction in cucumber after 12 h at room temperature (25 °C). The phage cocktail exhibited good antimicrobial efficiency, suggesting that it could reduce S. Typhimurium contamination of fresh produce. The strategy of developing cocktails of phages that target multiple host receptors can be used to develop novel biocontrol agents of S. Typhimurium.

Mutation of a Staphylococcus aureus temperate bacteriophage to a virulent one and evaluation of its application.

Bacteriophages have been suggested as alternative antimicrobial agents based on their host specificity and lytic activity. Therefore, it is necessary to obtain a virulent phage from a temperate one using molecular techniques to control Staphylococcus aureus efficiently. SA13, a novel temperate phage infecting S. aureus, was isolated and characterized. From this phage, mutant phages were generated by random deletion mutations, and a virulent mutant phage SA13m was selected. Comparative genome analysis revealed that the SA13m genome contains various nucleotide deletions in six genes encoding three hypothetical proteins and three lysogeny-associated proteins, including putative integrase, putative CI, and putative anti-repressor proteins. Mitomycin C induction of SA13m-resistant strains revealed that this mutant phage does not form lysogen, suggesting that SA13m is a virulent phage. In addition, SA13m showed rapid and long-lasting host cell growth inhibition activity. Furthermore, application of SA13m in sterilized milk showed that S. aureus was reduced to non-detectable levels both at refrigerator temperature (4 °C) and room temperature (25 °C), suggesting that SA13m can efficiently control the growth of S. aureus in foods. The virulent mutant phage SA13m could be used as a promising biocontrol agent against S. aureus without lysogen formation.

Noncanonical DNA-binding mode of repressor and its disassembly by antirepressor.

DNA-binding repressors are involved in transcriptional repression in many organisms. Disabling a repressor is a crucial step in activating expression of desired genes. Thus, several mechanisms have been identified for the removal of a stably bound repressor (Rep) from the operator. Here, we describe an uncharacterized mechanism of noncanonical DNA binding and induction by a Rep from the temperate Salmonella phage SPC32H; this mechanism was revealed using the crystal structures of homotetrameric Rep (92-198) and a hetero-octameric complex between the Rep and its antirepressor (Ant). The canonical method of inactivating a repressor is through the competitive binding of the antirepressor to the operator-binding site of the repressor; however, these studies revealed several noncanonical features. First, Ant does not compete for the DNA-binding region of Rep. Instead, the tetrameric Ant binds to the C-terminal domains of two asymmetric Rep dimers. Simultaneously, Ant facilitates the binding of the Rep N-terminal domains to Ant, resulting in the release of two Rep dimers from the bound DNA. Second, the dimer pairs of the N-terminal DNA-binding domains originate from different dimers of a Rep tetramer (trans model). This situation is different from that of other canonical Reps, in which two N-terminal DNA-binding domains from the same dimeric unit form a dimer upon DNA binding (cis model). On the basis of these observations, we propose a noncanonical model for the reversible inactivation of a Rep by an Ant.

Characterization and genome analysis of novel bacteriophages infecting the opportunistic human pathogens Klebsiella oxytoca and K. pneumoniae.

Klebsiella is a genus of well-known opportunistic human pathogens that are associated with diabetes mellitus and chronic pulmonary obstruction; however, this pathogen is often resistant to multiple drugs. To control this pathogen, two Klebsiella-infecting phages, K. oxytoca phage PKO111 and K. pneumoniae phage PKP126, were isolated from a sewage sample. Analysis of their host range revealed that they infect K. pneumoniae and K. oxytoca, suggesting host specificity for members of the genus Klebsiella. Stability tests confirmed that the phages are stable under various temperature (4 to 60 °C) and pH (3 to 11) conditions. A challenge assay showed that PKO111 and PKP126 inhibit growth of their host strains by 2 log and 4 log, respectively. Complete genome sequencing of the phages revealed that their genome sizes are quite different (168,758 bp for PKO111 and 50,934 bp for PKP126). Their genome annotation results showed that they have no human virulence-related genes, an important safety consideration. In addition, no lysogen-formation gene cluster was detected in either phage genome, suggesting that they are both virulent phages in their bacterial hosts. Based on these results, PKO111 and PKP126 may be good candidates for development of biocontrol agents against members of the genus Klebsiella for therapeutic purposes. A comparative analysis of tail-associated gene clusters of PKO111 and PKP126 revealed relatively low homology, suggesting that they might differ in the way they recognize and infect their specific hosts.

Characterization of a novel cell wall binding domain-containing Staphylococcus aureus endolysin LysSA97.

Endolysin from Staphylococcus aureus phage SA97 (LysSA97) was cloned and investigated. LysSA97 specifically lyse the staphylococcal strains and effectively disrupted staphylococcal biofilms. Bioinformatic analysis of LysSA97 revealed a novel putative cell wall binding domain (CBD) as well as two enzymatically active domains (EADs) containing cysteine, histidine-dependent amidohydrolases/peptidases (CHAP, PF05257) and N-acetylmuramoyl-L-alanine amidase (Amidase-3, PF01520) domains. Comparison of 98 endolysin genes of S. aureus phages deposited in GenBank showed that they can be classified into six groups based on their domain composition. Interestingly, approximately 80.61 % of the staphylococcal endolysins have a src-homology 3 (SH3, PF08460) domain as CBD, but the remaining 19.39 %, including LysSA97, has a putative C-terminal CBD with no homology to the known CBD. The fusion protein containing green fluorescent protein and the putative CBD of LysSA97 showed a specific binding spectrum against staphylococcal cells comparable to SH3 domain (PF08460), suggesting that the C-terminal domain of LysSA97 is a novel CBD of staphylococcal endolysins.

Characterization of a novel endolysin LysSA11 and its utility as a potent biocontrol agent against Staphylococcus aureus on food and utensils.

Here we show that the LysSA11 endolysin, derived from the virulent Staphylococcus aureus phage SA11, has lytic activity against staphylococcal strains. Bioinformatics analysis revealed an enzymatically active CHAP (cysteine, histidine-dependent amidohydrolases/peptidases) domain at the N-terminus of LysSA11 that showed amidase activity. A novel cell wall binding domain (CBD) in the C-terminus could bind to a broad spectrum of staphylococcal cells. The bactericidal activity of LysSA11 was determined in food and utensils artificially contaminated with methicillin-resistant S. aureus (MRSA). The amounts of MRSA bacteria in milk and on ham were significantly reduced by 1.44-log CFU/mL and 3.12-log CFU/cm3, respectively, within 15 min at refrigeration temperature (4 °C) and by 2.02-log CFU/mL and 3.37-log CFU/cm2, respectively, within 15 min at room temperature (25 °C). Moreover, a polypropylene plastic cutting board and a stainless steel knife artificially contaminated with approximately 4-log CFU/cm2 of MRSA also showed complete bacterial elimination after a 30-min treatment with 1.35 µM of LysSA11. The data presented here strongly suggest that the novel CBD-containing staphylococcal endolysin LysSA11 can be used both as a food antimicrobial and as a practical sanitizer for utensils.

A Novel Bacteriophage Targeting Cronobacter sakazakii Is a Potential Biocontrol Agent in Foods.

Cronobacter sakazakii is an important pathogen that causes high mortality in infants. Due to its occasional antibiotic resistance, a bacteriophage approach might be an alternative effective method for the control of this pathogen. To develop a novel biocontrol agent using bacteriophages, the C. sakazakii-infecting phage CR5 was newly isolated and characterized. Interestingly, this phage exhibited efficient and relatively durable host lysis activity. In addition, a specific gene knockout study and subsequent complementation experiment revealed that this phage infected the host strain using the bacterial flagella. The complete genome sequence analysis of phage CR5 showed that its genome contains 223,989 bp of DNA, including 231 predicted open reading frames (ORFs), and it has a G+C content of 50.06%. The annotated ORFs were classified into six functional groups (structure, packaging, host lysis, DNA manipulation, transcription, and additional functions); no gene was found to be related to virulence or toxin or lysogen formation, but >80% of the predicted ORFs are unknown. In addition, a phage proteomic analysis using SDS-PAGE and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) revealed that seven phage structural proteins are indeed present, supporting the ORF predictions. To verify the potential of this phage as a biocontrol agent against C. sakazakii, it was added to infant formula milk contaminated with a C. sakazakii clinical isolate or food isolate, revealing complete growth inhibition of the isolates by the addition of phage CR5 when the multiplicity of infection (MOI) was 10(5).

Plasmid-encoded MCP is involved in virulence, motility, and biofilm formation of Cronobacter sakazakii ATCC 29544.

The aim of this study was to elucidate the function of the plasmid-borne mcp (methyl-accepting chemotaxis protein) gene, which plays pleiotropic roles in Cronobacter sakazakii ATCC 29544. By searching for virulence factors using a random transposon insertion mutant library, we identified and sequenced a new plasmid, pCSA2, in C. sakazakii ATCC 29544. An in silico analysis of pCSA2 revealed that it included six putative open reading frames, and one of them was mcp. The mcp mutant was defective for invasion into and adhesion to epithelial cells, and the virulence of the mcp mutant was attenuated in rat pups. In addition, we demonstrated that putative MCP regulates the motility of C. sakazakii, and the expression of the flagellar genes was enhanced in the absence of a functional mcp gene. Furthermore, a lack of the mcp gene also impaired the ability of C. sakazakii to form a biofilm. Our results demonstrate a regulatory role for MCP in diverse biological processes, including the virulence of C. sakazakii ATCC 29544. To the best of our knowledge, this study is the first to elucidate a potential function of a plasmid-encoded MCP homolog in the C. sakazakii sequence type 8 (ST8) lineage.

Hfq plays important roles in virulence and stress adaptation in Cronobacter sakazakii ATCC 29544.

Cronobacter spp. are opportunistic pathogens that cause neonatal meningitis and sepsis with high mortality in neonates. Despite the peril associated with Cronobacter infection, the mechanisms of pathogenesis are still being unraveled. Hfq, which is known as an RNA chaperone, participates in the interaction with bacterial small RNAs (sRNAs) to regulate posttranscriptionally the expression of various genes. Recent studies have demonstrated that Hfq contributes to the pathogenesis of numerous species of bacteria, and its roles are varied between bacterial species. Here, we tried to elucidate the role of Hfq in C. sakazakii virulence. In the absence of hfq, C. sakazakii was highly attenuated in dissemination in vivo, showed defects in invasion (3-fold) into animal cells and survival (10(3)-fold) within host cells, and exhibited low resistance to hydrogen peroxide (10(2)-fold). Remarkably, the loss of hfq led to hypermotility on soft agar, which is contrary to what has been observed in other pathogenic bacteria. The hyperflagellated bacteria were likely to be attributable to the increased transcription of genes associated with flagellar biosynthesis in a strain lacking hfq. Together, these data strongly suggest that hfq plays important roles in the virulence of C. sakazakii by participating in the regulation of multiple genes.