Current and novel therapies for management of Acinetobacter baumannii-associated pneumonia.

Acinetobacter baumannii is a common pathogen associated with hospital-acquired pneumonia showing increased resistance to carbapenem and colistin antibiotics nowadays. Infections with A. baumannii cause high patient fatalities due to their capability to evade current antimicrobial therapies, emphasizing the urgency of developing viable therapeutics to treat A. baumannii-associated pneumonia. In this review, we explore current and novel therapeutic options for overcoming therapeutic failure when dealing with A. baumannii-associated pneumonia. Among them, antibiotic combination therapy administering several drugs simultaneously or alternately, is one promising approach for optimizing therapeutic success. However, it has been associated with inconsistent and inconclusive therapeutic outcomes across different studies. Therefore, it is critical to undertake additional clinical trials to ascertain the clinical effectiveness of different antibiotic combinations. We also discuss the prospective roles of novel antimicrobial therapies including antimicrobial peptides, bacteriophage-based therapy, repurposed drugs, naturally-occurring compounds, nanoparticle-based therapy, anti-virulence strategies, immunotherapy, photodynamic and sonodynamic therapy, for utilizing them as additional alternative therapy while tackling A. baumannii-associated pneumonia. Importantly, these innovative therapies further require pharmacokinetic and pharmacodynamic evaluation for safety, stability, immunogenicity, toxicity, and tolerability before they can be clinically approved as an alternative rescue therapy for A. baumannii-associated pulmonary infections.

Release of CM-12 from A2-type casein by the cleavage of Ser-Leu-Xaa at the C-terminus using Aspergillus oryzae alkaline protease.

BACKGROUND: Aspergillus oryzae protease can release the opioid peptide ß-casomorphin-10 (CM-10, YPFPGPIPNS, 60-69) from A2-type casein. However, not only is the yield of the active peptide low, but the key enzyme involved in processing has yet to be identified. RESULTS: A significant amount of the opioid peptide 60YPFPGPIPNSLP71 (CM-12) was produced from the A2-type casein peptide 53AQTQSLVYPFPGPIPNSLPQNIPPLTQTPV82 when the active protease in A. oryzae protease extract was fractionated with DEAE-Sepharose. The fractionated enzyme produced CM-12 from bovine A2-type casein but not from bovine A1 casein. A major protein of 34 kDa was purified and identified as an alkaline protease (Alp). Motif prediction of the Alp cleavage site using Multiple EM for Motif Elicitation analysis revealed preferable cleavage at the C-terminal end of Ser-Leu-Xaa for the release of CM-12. A2-type casein hydrolysate by Alp exhibited similar levels of opioid activity to that of synthetic CM-12 in cAMP-Glo assays with µ-opioid receptor-expressing HEK293 cells. These results suggest that CM-12 is a major opioid peptide in the casein hydrolysate. CONCLUSION: Our findings showed that Alp fractionated from A. oryzae protease extract produced the opioid peptide CM-12 from A2-type casein as a result of preferential cleavage at the C-terminal end of Ser-Leu-Xaa and the removal of coexisting enzymes. Moreover, docking predictions suggested a stable interaction between CM-12 and the 3D structure of Alp. Casein hydrolysate with Alp-containing CM-12 has the potential for use as a bioactive peptide material with opioid activity. © 2024 Society of Chemical Industry.

Enhanced tight junction in Caco-2 cells by the pretreatment with Lactobacillus johnsonii strain MG.

We investigated roles of Lactobacillus johnsonii MG (MG) isolated from mice with interaction with tight junction on gut barrier function with Caco-2 cell model. Pretreatment with MG enhanced barrier function and showed protective effect against Enterococcus faecium provided damage. MG treatment increased the gene expressions of transcriptional regulator NFKB and major tight junction protein, ZO-1.

Analysis host-recognition mechanism of staphylococcal kayvirus ɸSA039 reveals a novel strategy that protects Staphylococcus aureus against infection by Staphylococcus pseudintermedius Siphoviridae phages.

Following the emergence of antibiotic-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP), phage therapy has attracted significant attention as an alternative to antibiotic treatment. Bacteriophages belonging to kayvirus (previously known as Twort-like phages) have broad host range and are strictly lytic in Staphylococcus spp. Previous work revealed that kayvirus ɸSA039 has a host-recognition mechanism distinct from those of other known kayviruses: most of kayviruses use the backbone of wall teichoic acid (WTA) as their receptor; by contrast, ɸSA039 uses the ß-N-acetylglucosamine (ß-GlcNAc) residue in WTA. In this study, we found that ɸSA039 could switch its receptor to be able to infect S. aureus lacking the ß-GlcNAc residue by acquiring a spontaneous mutation in open reading frame (ORF) 100 and ORF102. Moreover, ɸSA039 could infect S. pseudintermedius, which has a different WTA structure than S. aureus. By comparison, with newly isolated S. pseudintermedius-specific phage (SP phages), we determined that glycosylation in WTA of S. pseudintermedius is essential for adsorption of SP phages, but not ɸSA039. Finally, we describe a novel strategy of S. aureus which protects the bacteria from infection of SP phages. Notably, glycosylation of ribitol phosphate (RboP) WTA by TarM or/and TarS prevents infection of S. aureus by SP phages. These findings could help to establish a new strategy for the treatment of S. aureus and S. pseudintermedius infection, as well as provide valuable insights into the biology of phage-host interactions.

Silviavirus phage ɸMR003 displays a broad host range against methicillin-resistant Staphylococcus aureus of human origin.

The emergence of life-threatening methicillin-resistant Staphylococcus aureus (MRSA) has led to increased interest in the use of bacteriophages as an alternative therapy to antibiotics. The success of phage therapy is greatly dependent on the selected phage possessing a wide host range. This study describes phage ɸMR003 isolated from sewage influent at a municipal wastewater treatment plant in Tokyo, Japan. ɸMR003 could infect 97% of 104 healthcare- and community-associated MRSA strains tested, compared with 73% for phage ɸSA012, which has a broad host range against bovine mastitis S. aureus. Genome analysis revealed that ɸMR003 belongs to the genus Silviavirus which has not been studied extensively. ɸMR003 recognizes and binds to wall teichoic acid (WTA) of S. aureus during infection. In silico comparisons of the genomes of ɸMR003 and ɸSA012 revealed that ORF117 and ORF119 of ɸMR003 are homologous to the putative receptor-binding proteins ORF103 and ORF105 of ɸSA012, with amino acid similarities of 75% and 72%, respectively. ORF104, which is an N-acetylglucosaminidase found in the ɸMR003 tail, may facilitate phage's infection onto the WTA-null S. aureus RN4220. The differences in tail and baseplate proteins may be key contributing factors to the different host specificities of ɸMR003 and ɸSA012. ɸMR003 showed strong adsorptivity, but not infectivity, against S. aureus SA003, which may be influenced by the bacterium's restriction modification system. This study expands our knowledge of the genomic diversity and host specificity of Silviavirus, which is a potential phage therapy candidate for MRSA infections.

Isolation, Characterisation and Complete Genome Sequence of a Tequatrovirus Phage, Escherichia phage KIT03, Which Simultaneously Infects Escherichia coli O157:H7 and Salmonella enterica.

Escherichia coli O157:H7 and Salmonella enterica subsp. enterica are the pathogens that frequently cause foodborne illness. Bacteriophage applications have been proposed as effective for preventing food contamination caused by these pathogenic bacteria. Escherichia phage KIT03 was isolated from the soil of a poultry farm in Kyoto, Japan. KIT03 can infect Escherichia coli O157:H7 and Salmonella enterica serotypes Choleraesuis and Enteritidis. One-step growth analysis revealed that KIT03 can propagate within its initial host (E. coli NBRC 3972), E. coli O157:H7 and S. Choleraesuis with an approximate burst size of 39, 51 and 37 phage particles per infected cell, respectively. The morphological type and genome annotation suggested that KIT03 belongs to the family Myoviridae, subfamily Tevenvirinae, genus Tequatrovirus. In vitro challenge tests demonstrated that KIT03 can lyse the tested bacteria and suppress their growth. Based on the susceptibility test and adsorption assay of KIT03 with E. coli K-12 BW25113 mutants, it was proposed that KIT03 may recognise and infect bacteria with a deficient outer core of lipopolysaccharides.

Analysis of phage resistance in Staphylococcus aureus SA003 reveals different binding mechanisms for the closely related Twort-like phages ɸSA012 and ɸSA039.

We have previously generated strains of Staphylococcus aureus SA003 resistant to its specific phage ɸSA012 through a long-term coevolution experiment. However, the DNA mutations responsible for the phenotypic change of phage resistance are unknown. Whole-genome analysis revealed eight genes that acquired mutations: six point mutations (five missense mutations and one nonsense mutation) and two deletions. Complementation of the phage-resistant strains by the wild-type alleles showed that five genes were linked to phage adsorption of ɸSA012, and two mutated host genes were linked to the inhibition of post-adsorption. Unlike ɸSA012, infection by ɸSA039, a close relative of ɸSA012, onto early coevolved phage-resistant SA003 (SA003R2) was impaired drastically. Here, we identified that ɸSA012 and ɸSA039 adsorb to the cell surface S. aureus SA003 through a different mechanism. ɸSA012 requires the backbone of wall teichoic acids (WTA), while ɸSA039 requires both backbone and the ß-GlcNAc residue. In silico analysis of the ɸSA039 genome revealed that several proteins in the tail and baseplate region were different from ɸSA012. The difference in tail and baseplate proteins might be the factor for specificity difference between ɸSA012 and ɸSA039.

Fate of Escherichia coli in dialysis device exposed into sewage influent and activated sludge.

Tracing the fate of pathogens in environmental water, particularly in wastewater, with a suitable methodology is a demanding task. We investigated the fate of Escherichia coli K12 in sewage influent and activated sludge using a novel approach that involves the application of a biologically stable dialysis device. The ion concentrations inside the device could reach that of surrounding solution when it was incubated in phosphate buffered saline for 2 h. E. coli K12 above 107 CFU mL-1 (inoculated in distilled water, influent, activated sludge) were introduced into the device and incubated in influent and activated sludge for 10 days. Without indigenous microorganisms, E. coli K12 could survive even with the limited ions and nutrients concentrations in influent and activated sludge. E. coli K12 abundance in influent and activated sludge were reduced by 60 and 85%, respectively, after just 1 day. The establishment of microbial community in wastewater played an important role in reducing E. coli K12. Bacteriophage propagated in filtered influent or activated sludge when E. coli K12 was introduced, but not in raw influent or activated sludge. The methodology developed in this study can be applied in the actual environmental water to trace the fate of pathogens.

The Presence of Two Receptor-Binding Proteins Contributes to the Wide Host Range of Staphylococcal Twort-Like Phages.

UNLABELLED: Thanks to their wide host range and virulence, staphylococcal bacteriophages (phages) belonging to the genus Twortlikevirus (staphylococcal Twort-like phages) are regarded as ideal candidates for clinical application for Staphylococcus aureus infections due to the emergence of antibiotic-resistant bacteria of this species. To increase the usability of these phages, it is necessary to understand the mechanism underlying host recognition, especially the receptor-binding proteins (RBPs) that determine host range. In this study, we found that the staphylococcal Twort-like phage ΦSA012 possesses at least two RBPs. Genomic analysis of five mutant phages of ΦSA012 revealed point mutations in orf103, in a region unique to staphylococcal Twort-like phages. Phages harboring mutated ORF103 could not infect S. aureus strains in which wall teichoic acids (WTAs) are glycosylated with α-N-acetylglucosamine (α-GlcNAc). A polyclonal antibody against ORF103 also inhibited infection by ΦSA012 in the presence of α-GlcNAc, suggesting that ORF103 binds to α-GlcNAc. In contrast, a polyclonal antibody against ORF105, a short tail fiber component previously shown to be an RBP, inhibited phage infection irrespective of the presence of α-GlcNAc. Immunoelectron microscopy indicated that ORF103 is a tail fiber component localized at the bottom of the baseplate. From these results, we conclude that ORF103 binds α-GlcNAc in WTAs, whereas ORF105, the primary RBP, is likely to bind the WTA backbone. These findings provide insight into the infection mechanism of staphylococcal Twort-like phages. IMPORTANCE: Staphylococcus phages belonging to the genus Twortlikevirus (called staphylococcal Twort-like phages) are considered promising agents for control of Staphylococcus aureus due to their wide host range and highly lytic capabilities. Although staphylococcal Twort-like phages have been studied widely for therapeutic purposes, the host recognition process of staphylococcal Twort-like phages remains unclear. This work provides new findings about the mechanisms of host recognition of the staphylococcal Twort-like phage ΦSA012. The details of the host recognition mechanism of ΦSA012 will allow us to analyze the mechanisms of infection and expand the utility of staphylococcal Twort-like phages for the control of S. aureus.

Persistence of antibiotic-resistant and -sensitive Proteus mirabilis strains in the digestive tract of the housefly (Musca domestica) and green bottle flies (Calliphoridae).

Synanthropic flies have been implicated in the rapid dissemination of antibiotic-resistant bacteria and resistance determinants in the biosphere. These flies stably harbor a considerable number of bacteria that exhibit resistance to various antibiotics, but the mechanisms underlying this phenomenon remain unclear. In this study, we investigated the persistence of antibiotic-resistant bacteria in the digestive tract of houseflies and green bottle flies, using Proteus mirabilis as a model microorganism. One resistant strain carried the blaTEM and aphA1 genes, and another carried a plasmid containing qnrD gene. Quantitative PCR and 454 pyrosequencing were used to monitor the relative abundance of the Proteus strains, as well as potential changes in the overall structure of the whole bacterial community incurred by the artificial induction of Proteus cultures. Both antibiotic-resistant and -sensitive P. mirabilis strains persisted in the fly digestive tract for at least 3 days, and there was no significant difference in the relative abundance of resistant and sensitive strains despite the lower growth rate of resistant strains when cultured in vitro. Therefore, conditions in the fly digestive tract may allow resistant strains to survive the competition with sensitive strains in the absence of antibiotic selective pressure. The composition of the fly-associated bacterial community changed over time, but the contribution of the artificially introduced P. mirabilis strains to these changes was not clear. In order to explain these changes, it will be necessary to obtain more information about bacterial interspecies antagonism in the fly digestive tract.

Seasonal variations in bacterial communities and antibiotic-resistant strains associated with green bottle flies (Diptera: Calliphoridae).

Green bottle flies occur frequently around human environments in Japan. Many species of green bottle flies have been studied with regard to their importance in forensic examinations or clinical therapies, but the bacterial communities associated with this group of flies have not been comprehensively investigated. In this research, 454 pyrosequencing was used to reveal the bacterial communities in green bottle flies collected in different seasons. Meanwhile, the bacteria were screened with selective media and tested for antibiotic susceptibility. Samples collected in three different seasons harbored distinctive bacterial communities. The predominant genera associated with green bottles flies were Staphylococcus in spring, Ignatzschineria in summer, and Vagococcus, Dysgonomonas, and an unclassified Acetobacteraceae in autumn. An upward trend in bacterial community diversity was observed from spring to autumn. Changes in climatic conditions could be the cause of these seasonal variations in fly-associated bacterial communities. The species of isolated antibiotic-resistant bacteria also differed across seasons, but it was difficult to correlate seasonal changes in antibiotic-resistant bacteria with changes in whole communities. A number of multiple-antibiotic-resistant bacteria were isolated, and some of these strains were closely affiliated with pathogens such as Enterococcus faecalis and Enterococcus faecium, which could cause serious threats to public health. Overall, this research provided us with information about the composition and seasonality of bacterial communities in green bottle flies, and highlighted the risks of fly-mediated dissemination of antibiotic-resistant pathogens.

Selective bacteriophages reduce the emergence of resistant bacteria in bacteriophage-antibiotic combination therapy.

Escherichia coli O157:H7 is a globally important foodborne pathogen with implications for food safety. Antibiotic treatment for O157 may potentially contribute to the exacerbation of hemolytic uremic syndrome, and the increasing prevalence of antibiotic-resistant strains necessitates the development of new treatment strategies. In this study, the bactericidal effects and resistance development of antibiotic and bacteriophage monotherapy were compared with those of combination therapy against O157. Experiments involving continuous exposure of O157 to phages and antibiotics, along with genetic deletion studies, revealed that the deletion of glpT and uhpT significantly increased resistance to fosfomycin. Furthermore, we found that OmpC functions as a receptor for the PP01 phage, which infects O157, and FhuA functions as a receptor for the newly isolated SP15 phage, targeting O157. In the glpT and uhpT deletion mutants, additional deletion in ompC, the receptor for the PP01 phage, increased resistance to fosfomycin. These findings suggest that specific phages may contribute to antibiotic resistance by selecting the emergence of gene mutations responsible for both phage and antibiotic resistance. While combination therapy with phages and antibiotics holds promise for the treatment of bacterial infections, careful consideration of phage selection is necessary.IMPORTANCEThe combination treatment of fosfomycin and bacteriophages against Escherichia coli O157 demonstrated superior bactericidal efficacy compared to monotherapy, effectively suppressing the emergence of resistance. However, mutations selected by phage PP01 led to enhanced resistance not only to the phage but also to fosfomycin. These findings underscore the importance of exercising caution in selecting phages for combination therapy, as resistance selected by specific phages may increase the risk of developing antibiotic resistance.

Metabolic remodeling by RNA polymerase gene mutations is associated with reduced ß-lactam susceptibility in oxacillin-susceptible MRSA.

The emergence of oxacillin-susceptible methicillin-resistant Staphylococcus aureus (OS-MRSA) has imposed further challenges to the clinical management of MRSA infections. When exposed to ß-lactam antibiotics, these strains can easily acquire reduced ß-lactam susceptibility through chromosomal mutations, including those in RNA polymerase (RNAP) genes such as rpoBC, which may then lead to treatment failure. Despite the increasing prevalence of such strains and the apparent challenges they pose for diagnosis and treatment, there is limited information available on the actual mechanisms underlying such chromosomal mutation-related transitions to reduced ß-lactam susceptibility, as it does not directly associate with the expression of mecA. This study investigated the cellular physiology and metabolism of six missense mutants with reduced oxacillin susceptibility, each carrying respective mutations on RpoBH929P, RpoBQ645H, RpoCG950R, RpoCG498D, RpiAA64E, and FruBA211E, using capillary electrophoresis-mass spectrometry-based metabolomics analysis. Our results showed that rpoBC mutations caused RNAP transcription dysfunction, leading to an intracellular accumulation of ribonucleotides. These mutations also led to the accumulation of UDP-Glc/Gal and UDP-GlcNAc, which are precursors of UTP-associated peptidoglycan and wall teichoic acid. Excessive amounts of building blocks then contributed to the cell wall thickening of mutant strains, as observed in transmission electron microscopy, and ultimately resulted in decreased susceptibility to ß-lactam in OS-MRSA. IMPORTANCE: The emergence of oxacillin-susceptible methicillin-resistant Staphylococcus aureus (OS-MRSA) strains has created new challenges for treating MRSA infections. These strains can become resistant to ß-lactam antibiotics through chromosomal mutations, including those in the RNA polymerase (RNAP) genes such as rpoBC, leading to treatment failure. This study investigated the mechanisms underlying reduced ß-lactam susceptibility in four rpoBC mutants of OS-MRSA. The results showed that rpoBC mutations caused RNAP transcription dysfunction, leading to an intracellular accumulation of ribonucleotides and precursors of peptidoglycan as well as wall teichoic acid. This, in turn, caused thickening of the cell wall and ultimately resulted in decreased susceptibility to ß-lactam in OS-MRSA. These findings provide insights into the mechanisms of antibiotic resistance in OS-MRSA and highlight the importance of continued research in developing effective treatments to combat antibiotic resistance.

Efficient synthesis of CRISPR-Cas13a-antimicrobial capsids against MRSA facilitated by silent mutation incorporation.

In response to the escalating global threat of antimicrobial resistance, our laboratory has established a phagemid packaging system for the generation of CRISPR-Cas13a-antimicrobial capsids targeting methicillin-resistant Staphylococcus aureus (MRSA). However, a significant challenge arose during the packaging process: the unintentional production of wild-type phages alongside the antimicrobial capsids. To address this issue, the phagemid packaging system was optimized by strategically incorporated silent mutations. This approach effectively minimized contamination risks without compromising packaging efficiency. The study identified the indispensable role of phage packaging genes, particularly terL-terS, in efficient phagemid packaging. Additionally, the elimination of homologous sequences between the phagemid and wild-type phage genome was crucial in preventing wild-type phage contamination. The optimized phagemid-LSAB(mosaic) demonstrated sequence-specific killing, efficiently eliminating MRSA strains carrying target antibiotic-resistant genes. While acknowledging the need for further exploration across bacterial species and in vivo validation, this refined phagemid packaging system offers a valuable advancement in the development of CRISPR-Cas13a-based antimicrobials, shedding light on potential solutions in the ongoing battle against bacterial infections.

Exploring indoor and outdoor dust as a potential tool for detection and monitoring of COVID-19 transmission.

This study investigated the potential of using SARS-CoV-2 viral concentrations in dust as an additional surveillance tool for early detection and monitoring of COVID-19 transmission. Dust samples were collected from 8 public locations in 16 districts of Bangkok, Thailand, from June to August 2021. SARS-CoV-2 RNA concentrations in dust were quantified, and their correlation with community case incidence was assessed. Our findings revealed a positive correlation between viral concentrations detected in dust and the relative risk of COVID-19. The highest risk was observed with no delay (0-day lag), and this risk gradually decreased as the lag time increased. We observed an overall decline in viral concentrations in public places during lockdown, closely associated with reduced human mobility. The effective reproduction number for COVID-19 transmission remained above one throughout the study period, suggesting that transmission may persist in locations beyond public areas even after the lockdown measures were in place.

Comparative analysis of bacterial community and antibiotic-resistant strains in different developmental stages of the housefly (Musca domestica).

The housefly (Musca domestica) is an important host for a variety of bacteria, including some pathogenic and antibiotic-resistant strains. To further investigate the relationship between the housefly and the bacteria it harbors, it is necessary to understand the fate of microorganisms during the larval metamorphosis. The major bacterial communities in three developmental stages of the housefly (maggot, pupa, and adult fly) were investigated by a culture-independent method, polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis of 16S rRNA genes. The bacteria that were identified using DGGE analysis spanned phyla Proteobacteria, Firmicutes, and Bacteroidetes. Changes in the predominant genera were observed during the housefly development. Bacteroides, Koukoulia, and Schineria were detected in maggots, Neisseria in pupae, and Macrococcus, Lactococcus, and Kurthia in adult flies. Antibiotic-resistant bacteria were screened using a selective medium and tested for antibiotic susceptibility. Most resistant isolates from maggots and pupae were classified as Proteus spp., while those from adult flies were much more diverse and spanned 12 genera. Among 20 tested strains across the three stages, 18 were resistant to at least two antibiotics. Overall, we demonstrated that there are changes in the major bacterial communities and antibiotic-resistant strains as the housefly develops.

Iodine from bacterial iodide oxidization by Roseovarius spp. inhibits the growth of other bacteria.

Microbial activities in brine, seawater, or estuarine mud are involved in iodine cycle. To investigate the effects of the microbiologically induced iodine on other bacteria in the environment, a total of 13 bacteria that potentially participated in the iodide-oxidizing process were isolated from water or biofilm at a location containing 131 µg ml(-1) iodide. Three distinct strains were further identified as Roseovarius spp. based on 16 S rRNA gene sequences after being distinguished by restriction fragment length polymorphism analysis. Morphological characteristics of these three Roseovarius spp. varied considerably across and within strains. Iodine production increased with Roseovarius spp. growth when cultured in Marine Broth with 200 µg ml(-1) iodide (I(-)). When 10(6) CFU/ml Escherichia coli, Pseudomonas aeruginosa, and Bacillus pumilus were exposed to various concentrations of molecular iodine (I(2)), the minimum inhibitory concentrations (MICs) were 0.5, 1.0, and 1.0 µg ml(-1), respectively. However, fivefold increases in the MICs for Roseovarius spp. were obtained. In co-cultured Roseovarius sp. IOB-7 and E. coli in Marine Broth containing iodide (I(-)), the molecular iodine concentration was estimated to be 0.76 µg ml(-1) after 24 h and less than 50 % of E. coli was viable compared to that co-cultured without iodide. The growth inhibition of E. coli was also observed in co-cultures with the two other Roseovarius spp. strains when the molecular iodine concentration was assumed to be 0.52 µg ml(-1).

Immunomodulatory effects of extracellular glyceraldehyde 3-phosphate dehydrogenase of exopolysaccharide-producing Lactiplantibacillus plantarum JCM 1149.

Probiotic lactic acid bacteria evoke immunomodulatory effects in the host; however, the reasons for the different effects of various species and strains remain to be elucidated. To clarify the critical immunomodulatory components and impact of exopolysaccharide (EPS) in Lactiplantibacillus plantarum, 11 types of L. plantarum strains were compared for the production of EPS, inflammatory cytokines, interleukin-6 and -12, and the anti-inflammatory cytokine, interleukin-10, from THP-1 differentiated dendritic cells. EPS in the fermented medium correlated with cytokine-inducing activities. L. plantarum JCM 1149, with the highest production of EPS, also induced interleukin-6, -10, and -12 among the 11 tested strains. Notably, the cytokine-producing activities overlapped with the protein fraction in gel filtration chromatography but not with EPS, which has been reported to exert immunomodulatory effects. The 41 kDa protein that coexisted with EPS was purified as a major active component and identified as glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a known moonlighting protein. GAPDH secretion was reduced when EPS synthesis inhibitors were added to the culture medium. RNA sequencing of GAPDH-treated THP-1 cells revealed an up-regulation in the expression of genes involved in transcriptional regulation, cell surface receptor signalling, immune response, and matrix components. Here, we report, to our knowledge for the first time, that the cell surface-associated L. plantarum GAPDH plays a crucial role in cytokine production in THP-1 cells, but EPS with less activity may help GAPDH secretion.

GroEL Secreted from Bacillus subtilis Natto Exerted a Crucial Role for Anti-Inflammatory IL-10 Induction in THP-1 Cells.

Although diverse immunomodulatory reactions of probiotic bacteria have been reported, this effect via Bacillus subtilis natto remains unclear, despite its long consumption history in Japan and usage in Natto production. Hence, we performed a comparative analysis of the immunomodulatory activities of 23 types of B. subtilis natto isolated from Natto products to elucidate the key active components. Among the isolated 23 strains, the supernatant from B. subtilis strain 1 fermented medium showed the highest induction of anti-inflammatory IL-10 and pro-inflammatory IL-12 in THP-1 dendritic cells (THP-1 DC) after co-incubation. We isolated the active component from strain 1 cultured medium and employed DEAE-Sepharose chromatography with 0.5 M NaCl elution for fractionation. IL-10-inducing activity was specific to an approximately 60 kDa protein, GroEL, which was identified as a chaperone protein and was significantly reduced with anti-GroEL antibody. Differential expression analysis of strains 1 and 15, which had the lowest cytokine-producing activity, showed a higher expression of various genes involved in chaperones and sporulation in strain 1. Furthermore, GroEL production was induced in spore-forming medium. The present study is the first to show that the chaperone protein GroEL, secreted by B. subtilis natto during sporulation, plays a crucial role in IL-10 and IL-12 production in THP-1 DC.

Uptake of Levilactobacillus brevis JCM 1059 by THP-1 Cells via Interaction between SlpB and CAP-1 Promotes Cytokine Production.

Several probiotic lactic acid bacteria (LAB) exert immunomodulatory effects on the host. However, the reasons for the different effects of LAB have not been fully elucidated. To understand the different immunomodulatory effects of LAB, we evaluated the levels of critical molecules in differentiated monocytic THP-1 and dendritic cells (DCs) following the uptake of various LAB strains. Lactobacillus helveticus JCM 1120, Lactobacillus acidophilus JCM 1132, Levilactobacillus brevis JCM 1059, and Lentilactobacillus kefiri JCM 5818 showed significantly higher uptake among the 12 LAB species tested. The uptake of microbeads by THP-1 DC increased when coupled with the surface layer proteins (Slps) from the tested strains. SlpB was mainly observed in the L. brevis JCM 1059 Slps extract. The expected cell surface receptor for SlpB on THP-1 DC was purified using SlpB-coupled affinity resin and identified as adenylyl cyclase-associated protein 1 (CAP-1). SlpB binding to THP-1 DC decreased after the addition of anti-CAP-1 and anti-DC-SIGN antibodies but not after the addition of anti-macrophage-inducible C-type lectin (Mincle) antibody. These results suggest that SlpB on L. brevis JCM 1059 plays preferentially binds to CAP-1 on THP-1 DC and plays a crucial role in bacterial uptake by THP-1 cells as well as in subsequent interleukin-12 (IL-12) production.