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Kazachstania slooffiae is a dimorphic fungus which colonizes the feces and gastrointestinal tract of postweaning pigs. This fungus persists in the gut environment of piglets into adulthood and is implicated in porcine health through microbe-microbe and microbe-host interactions. Here, we report a draft genome sequence for K. slooffiae ABBL.

The increasing spread of antibiotic-resistant microorganisms has led to the necessity of developing alternative antimicrobial treatments. The use of peptidoglycan hydrolases is a promising approach to combat bacterial infections. In our study, we constructed a 2 kb-triple-acting fusion gene (TF) encoding the N-terminal amidase-5 domain of streptococcal LambdaSA2 prophage endolysin (D-glutamine-L-lysin endopeptidase), a mid-protein amidase-2 domain derived from the staphylococcal phage 2638A endolysin (N-acetylmuramoyl-L-alanine amidase) and the mature version (246 residues) of the Staphylococcus simulans Lysostaphin bacteriocin (glycyl-glycine endopeptidase) at the C-terminus. The TF gene was expressed in Nicotiana benthamiana plants using the non-replicating Cowpea mosaic virus (CPMV)-based vector pEAQ-HT and the replicating Alternanthera mosaic virus (AltMV)-based pGD5TGB1L8823-MCS-CP3 vector, and in Escherichia coli using pET expression vectors pET26b+ and pET28a+. The resulting poor expression of this fusion protein in plants prompted the construction of a TF gene codon-optimized for expression in tobacco plants, resulting in an improved codon adaptation index (CAI) from 0.79 (TF gene) to 0.93 (TFnt gene). Incorporation of the TFnt gene into the pEAQ-HT vector, followed by transient expression in N. benthamiana, led to accumulation of TFnt to an approximate level of 0.12 mg/g of fresh leaf weight. Antimicrobial activity of purified plant- and bacterial-produced TFnt proteins was assessed against two strains of Gram-positive Staphylococcus aureus 305 and Newman. The results showed that plant-produced TFnt protein was preferentially active against S. aureus 305, showing 14% of growth inhibition, while the bacterial-produced TFnt revealed significant antimicrobial activity against both strains, showing 68 (IC50 25 µg/ml) and 60% (IC50 71 µg/ml) growth inhibition against S. aureus 305 and Newman, respectively. Although the combination of codon optimization and transient expression using the non-replicating pEAQ-HT expression vector facilitated production of the TFnt protein in plants, the most functionally active antimicrobial protein was obtained using the prokaryotic expression system.

Kazachstania slooffiae is a fungus commonly isolated from the gastrointestinal tract and feces of post-weaning pigs. Studies have implicated its ability to positively alter piglet gut health through potential symbioses with beneficial bacteria, including Lactobacillus and Prevotella, in providing amino acids as an energy source for microbial and piglet growth, and it has been found to be positively correlated with short-chain fatty acids in the piglet gut. However, basic mycological information remains limited, hampering in vitro studies. In this study, we characterized the growth parameters, biofilm formation ability, susceptibility to antimicrobials, and genetic relatedness of K. slooffiae to other fungal isolates. Optimal fungal growth conditions were determined, no antifungal resistance was found against multiple classes of antifungal drugs (azoles, echinocandins, polyenes, or pyrimidine analogues), and dimorphic growth was observed. K. slooffiae produced biofilms that became more complex in the presence of Lactobacillus acidophilus supernatant, suggesting positive interactions with this bacterium in the gut, while Enterococcus faecalis supernatant decreased density, suggesting an antagonistic interaction. This study characterizes the in vitro growth conditions that are optimal for further studies of K. slooffiae, which is an important step in defining the role and interactions of K. slooffiae in the porcine gut environment.

Weaning is a period of environmental changes and stress that results in significant alterations to the piglet gut microbiome and is associated with a predisposition to disease, making potential interventions of interest to the swine industry. In other animals, interactions between the bacteriome and mycobiome can result in altered nutrient absorption and susceptibility to disease, but these interactions remain poorly understood in pigs. Recently, we assessed the colonization dynamics of fungi and bacteria in the gastrointestinal tract of piglets at a single time point post-weaning (day 35) and inferred interactions were found between fungal and bacterial members of the porcine gut ecosystem. In this study, we performed a longitudinal assessment of the fecal bacteriome and mycobiome of piglets from birth through the weaning transition. Piglet feces in this study showed a dramatic shift over time in the bacterial and fungal communities, as well as an increase in network connectivity between the two kingdoms. The piglet fecal bacteriome showed a relatively stable and predictable pattern of development from Bacteroidaceae to Prevotellaceae, as seen in other studies, while the mycobiome demonstrated a loss in diversity over time with a post-weaning population dominated by Saccharomycetaceae. The mycobiome demonstrated a more transient community that is likely driven by factors such as diet or environmental exposure rather than an organized pattern of colonization and succession evidenced by fecal sample taxonomic clustering with nursey feed samples post-weaning. Due to the potential tractability of the community, the mycobiome may be a viable candidate for potential microbial interventions that will alter piglet health and growth during the weaning transition.

The increasing spread of antibiotic-resistant pathogens has raised the interest in alternative antimicrobial treatments. In our study, the functionally active gram-negative bacterium bacteriophage CP933 endolysin was produced in Nicotiana benthamiana plants by a combination of transient expression and vacuole targeting strategies, and its antimicrobial activity was investigated. Expression of the cp933 gene in E. coli led to growth inhibition and lysis of the host cells or production of trace amounts of CP933. Cytoplasmic expression of the cp933 gene in plants using Potato virus X-based transient expression vectors (pP2C2S and pGR107) resulted in death of the apical portion of experimental plants. To protect plants against the toxic effects of the CP933 protein, the cp933 coding region was fused at its Nterminus to an N-terminal signal peptide from the potato proteinase inhibitor I to direct CP933 to the delta-type vacuoles. Plants producing the CP933 fusion protein did not exhibit the severe toxic effects seen with the unfused protein and the level of expression was 0.16 mg/g of plant tissue. Antimicrobial assays revealed that, in contrast to gram-negative bacterium E. coli (BL21(DE3)), the gram-positive plant pathogenic bacterium Clavibacter michiganensis was more susceptible to the plant-produced CP933, showing 18% growth inhibition. The results of our experiments demonstrate that the combination of transient expression and protein targeting to the delta vacuoles is a promising approach to produce functionally active proteins that exhibit toxicity when expressed in plant cells.

Antibiotic resistance and the colonization of bacteria on surfaces, often as biofilms, prolong hospitalization periods, increase mortality, and are thus major concerns for health care providers. There is an urgent need for antimicrobial and antibiofilm surface treatments that are permanent, can eradicate both biofilms and planktonic pathogens over long periods of time, and do not select for resistant strains. In this study, we have demonstrated a simple, robust, and biocompatible method that utilizes the adhesive property of polydopamine (PDA) to covalently attach the antimicrobial enzyme lysostaphin (Lst) to a variety of surfaces to generate antibacterial and antibiofilm interfaces. The immobilization of the recombinant Lst onto PDA-coated surfaces was carried out under physiological conditions, most probably through the C-terminal His6-tag fragment of the enzyme, minimizing the losses of bioagent activity. The modified surfaces were extensively characterized by X-ray photoelectron spectroscopy and peak force quantitative nanomechanical mapping (PeakForce QNM) AFM-based method, and the presence of Lst on the surfaces was further confirmed immunochemically using anti-Lst antibody. We also found that, in contrast to the physically adsorbed Lst, the covalently attached Lst does not leach from the surfaces and maintains its endopeptidase activity to degrade the staphylococcal cell wall, avoiding most intracellular bacterial resistance mechanisms. Moreover, the Lst-coated surfaces kill hospital strains of Staphylococcus aureus in less than 15 min and prevent biofilm formation. This immobilization method should be applicable also to other proteins and enzymes that are recombinantly expressed to include the His6-tag fragment.

Multi-drug resistant bacteria are a persistent problem in modern health care, food safety and animal health. There is a need for new antimicrobials to replace over used conventional antibiotics. Here we describe engineered triple-acting staphylolytic peptidoglycan hydrolases wherein three unique antimicrobial activities from two parental proteins are combined into a single fusion protein. This effectively reduces the incidence of resistant strain development. The fusion protein reduced colonization by Staphylococcus aureus in a rat nasal colonization model, surpassing the efficacy of either parental protein. Modification of a triple-acting lytic construct with a protein transduction domain significantly enhanced both biofilm eradication and the ability to kill intracellular S. aureus as demonstrated in cultured mammary epithelial cells and in a mouse model of staphylococcal mastitis. Interestingly, the protein transduction domain was not necessary for reducing the intracellular pathogens in cultured osteoblasts or in two mouse models of osteomyelitis, highlighting the vagaries of exactly how protein transduction domains facilitate protein uptake. Bacterial cell wall degrading enzyme antimicrobials can be engineered to enhance their value as potent therapeutics., Scientific Reports, 6, ISSN:2045-2322

Staphylococcus aureus is a notorious pathogen highly successful at developing resistance to virtually all antibiotics to which it is exposed. Staphylococcal phage 2638A endolysin is a peptidoglycan hydrolase that is lytic for S. aureus when exposed externally, making it a new candidate antimicrobial. It shares a common protein organization with more than 40 other reported staphylococcal peptidoglycan hydrolases. There is an N-terminal M23 peptidase domain, a mid-protein amidase 2 domain (N-acetylmuramoyl-L-alanine amidase), and a C-terminal SH3b cell wall-binding domain. It is the first phage endolysin reported with a secondary translational start site in the inter-lytic-domain region between the peptidase and amidase domains. Deletion analysis indicates that the amidase domain confers most of the lytic activity and requires the full SH3b domain for maximal activity. Although it is common for one domain to demonstrate a dominant activity over the other, the 2638A endolysin is the first in this class of proteins to have a high-activity amidase domain (dominant over the N-terminal peptidase domain). The high activity amidase domain is an important finding in the quest for high-activity staphylolytic domains targeting novel peptidoglycan bonds.

Staphylococcal peptidoglycan hydrolases are a potential new source of antimicrobials. A large subset harbors C-terminal SH3b_5 cell wall binding domains. These C-terminal domains have been shown to be necessary for accurate cell wall recognition and subsequent staphylolytic activity for some endolysins. Over fifty proteins of staphylococcal or phage origin containing SH3b domains were aligned, yielding five highly repetitive groups of proteins. Representative C-termini from these five groups, and six staphylococcal proteins for which no homologues have been identified, were aligned, revealing two distinct SH3b_5 subgroups with overlapping but differentially conserved residues. A premise behind this research is that there may be unique cell wall binding properties conferred by these staphylococcal domains that could be exploited to specifically enhance anti-staphylococcal efficacy in heterologous protein fusion constructs. To identify functional differences between the two subgroups, the native Cpl-7 cell wall binding domains of the streptococcal LambdaSa2 endolysin were replaced by staphylococcal SH3b domains from both subgroups. SH3b domains from either lysostaphin (bacteriocin) or LysK (phage endolysin) resulted in a approximately 5x increase in staphylolytic activity conferred on the streptococcal endopeptidase domain, and surprisingly these same fusions maintained significant streptolytic activity suggesting that the staphylococcal SH3b domains are not always staphylococcal-specific. A comparison of the differences in lytic activity conferred on the LambdaSa2 endopeptidase domain by either LysK or lysostaphin SH3b domain differed by no more than a factor of two. Through the collection of peptidoglycan hydrolase sequences, three new putative intron-containing phage endolysin genes were identified in public data sets for the phages G1, X2 and 85.

The Staphylococcus aureus bacteriophage phi11 endolysin has two peptidoglycan hydrolase domains (endopeptidase and amidase) and an SH3b cell wall-binding domain. In turbidity reduction assays, the purified protein can lyse untreated staphylococcal mastitis pathogens, Staphylococcus aureus and coagulase-negative staphylococci (Staphylococcus chronogenes, Staphylococcus epidermidis, Staphylococcus hyicus, Staphylococcus simulans, Staphylococcus warneri and Staphylococcus xylosus), making it a strong candidate protein antimicrobial. This lytic activity is maintained at the pH (6.7), and the "free" calcium concentration (3 mM) of milk. Truncated endolysin-derived proteins containing only the endopeptidase domain also lyse staphylococci in the absence of the SH3b-binding domain.

Journal of Antimicrobial Chemotherapy, 70 (5), ISSN:0305-7453, ISSN:1460-2091

Peptidoglycan hydrolases are an effective new source of antimicrobials. A chimeric fusion protein of the Ply187 endopeptidase domain and LysK SH3b cell wall–binding domain is a potent agent against Staphylococcus aureus in four functional assays.

LysK is a staphylococcal bacteriophage endolysin composed of three domains: an N-terminal cysteine, histidine-dependent amidohydrolases/peptidases (CHAP) endopeptidase domain, a midprotein amidase 2 domain, and a C-terminal SH3b_5 (SH3b) cell wall-binding domain. Both catalytic domains are active on purified peptidoglycan by positive-ion electrospray ionization MS. The cut sites are identical to LytA (phi11 endolysin), with cleavage between d-alanine of the stem peptide and glycine of the cross-bridge peptide, and N-acetylmuramoyl-l-alanine amidase activity. Truncations of the LysK containing just the CHAP domain lyse Staphylococcus aureus cells in zymogram analysis, plate lysis, and turbidity reduction assays but have no detectable activity in a minimal inhibitory concentration (MIC) assay. In contrast, truncations harboring just the amidase lytic domain show faint activity in both the zymogram and turbidity reduction assays, but no detectable activity in either plate lysis or MIC assays. A fusion of the CHAP domain to the SH3b domain has near full-length LysK lytic activity, suggesting the need for a C-terminal binding domain. Both LysK and the CHAP-SH3b fusion were shown to lyse untreated S. aureus and the coagulase-negative strains. In the checkerboard assay, the CHAP-SH3b fusion achieves the same level of antimicrobial synergy with lysostaphin as the full-length LysK.

LysK is the endolysin from the staphylococcal bacteriophage K, and can digest the cell wall of many staphylococci. Lysostaphin is a bacteriocin secreted by Staphylococcus simulans to kill Staphylococcus aureus. Both LysK and lysostaphin have been shown to lyse methicillin-resistant S. aureus (MRSA). This study describes optimal reaction conditions for the recombinant His-tagged LysK protein (pH range pH 6-10, and 0.3-0.5 M NaCl), and C-His-LysK MIC (32.85+/-4.87 mug mL(-1)). LysK and lysostaphin demonstrate antimicrobial synergy by the checkerboard assay.

Streptococcal pathogens contribute to a wide variety of human and livestock diseases. The routine use of antibiotics to battle these pathogens has produced a new class of multidrug-resistant streptococci. Thus, there is a need for new antimicrobials. Bacteriophage endolysins (peptidoglycan hydrolases) comprise one group of new antimicrobials that are reportedly refractory to resistance development. The LambdaSa2 prophage endolysin gene was recently isolated from a Group B streptococcal genome, expressed on an Escherichia coli plasmid, and shown by homology screening and biochemical analysis to harbor an amidase-5 (endopeptidase) domain, an amidase-4 (glycosidase) domain, and two Cpl-7 cell wall-binding domains. In this study, turbidity reduction and plate lysis assays indicate that this hydrolase shows strong lytic activity toward Streptococcus pyogenes, Streptococcus dysgalactiae, Streptococcus uberis, Streptococcus equi, GES, and GGS. Deletion analysis indicates that the N-terminal endopeptidase domain with both Cpl-7 domains can lyse with a higher specific activity than the full-length protein (against some strains). This dual Cpl-7 domain truncated version also shows weak lytic activity against methicillin-resistant Staphylococcus aureus (MRSA) and the coagulase negative staphylococci, Staphylococcus xylosus. The truncated constructs harboring the glycosidase domain are virtually inactive, showing only minimal activity on plate lysis assays.