The peritoneal cavity (PerC) is a unique compartment within which a variety of immune cells reside, and from which macrophages (MØ) are commonly drawn for functional studies. Here we define two MØ ...subsets that coexist in PerC in adult mice. One, provisionally called the large peritoneal MØ (LPM), contains approximately 90% of the PerC MØ in unstimulated animals but disappears rapidly from PerC following lipopolysaccharide (LPS) or thioglycolate stimulation. These cells express high levels of the canonical MØ surface markers, CD11b and F4/80. The second subset, referred to as small peritoneal MØ (SPM), expresses substantially lower levels of CD11b and F4/80 but expresses high levels of MHC-II, which is not expressed on LPM. SPM, which predominates in PerC after LPS or thioglycolate stimulation, does not derive from LPM. Instead, it derives from blood monocytes that rapidly enter the PerC after stimulation and differentiate to mature SPM within 2 to 4 d. Both subsets show clear phagocytic activity and both produce nitric oxide (NO) in response to LPS stimulation in vivo. However, their responses to LPS show key differences: in vitro, LPS stimulates LPM, but not SPM, to produce NO; in vivo, LPS stimulates both subsets to produce NO, albeit with different response patterns. These findings extend current models of MØ heterogeneity and shed new light on PerC MØ diversity, development, and function. Thus, they introduce a new context for interpreting (and reinterpreting) data from ex vivo studies with PerC MØ.
Clostridium difficile is a leading cause of nosocomial infections worldwide and has become an urgent public health threat requiring immediate attention. Epidemic lineages of the BI/NAP1/027 strain ...type have emerged and spread through health care systems across the globe over the past decade. Limiting person-to-person transmission and eradicating C. difficile, especially the BI/NAP1/027 strain type, from health care facilities are difficult due to the abundant shedding of spores that are impervious to most interventions. Effective prophylaxis for C. difficile infection (CDI) is lacking. We have genetically modified a contractile R-type bacteriocin ("diffocin") from C. difficile strain CD4 to kill BI/NAP1/027-type strains for this purpose. The natural receptor binding protein (RBP) responsible for diffocin targeting was replaced with a newly discovered RBP identified within a prophage of a BI/NAP1/027-type target strain by genome mining. The resulting modified diffocins (a.k.a. Avidocin-CDs), Av-CD291.1 and Av-CD291.2, were stable and killed all 16 tested BI/NAP1/027-type strains. Av-CD291.2 administered in drinking water survived passage through the mouse gastrointestinal (GI) tract, did not detectably alter the mouse gut microbiota or disrupt natural colonization resistance to C. difficile or the vancomycin-resistant Enterococcus faecium (VREF), and prevented antibiotic-induced colonization of mice inoculated with BI/NAP1/027-type spores. Given the high incidence and virulence of the pathogen, preventing colonization by BI/NAP1/027-type strains and limiting their transmission could significantly reduce the occurrence of the most severe CDIs. This modified diffocin represents a prototype of an Avidocin-CD platform capable of producing targetable, precision anti-C. difficile agents that can prevent and potentially treat CDIs without disrupting protective indigenous microbiota.
Treatment and prevention strategies for bacterial diseases rely heavily on traditional antibiotics, which impose strong selection for resistance and disrupt protective microbiota. One consequence has been an upsurge of opportunistic pathogens, such as Clostridium difficile, that exploit antibiotic-induced disruptions in gut microbiota to proliferate and cause life-threatening diseases. We have developed alternative agents that utilize contractile bactericidal protein complexes (R-type bacteriocins) to kill specific C. difficile pathogens. Efficacy in a preclinical animal study indicates these molecules warrant further development as potential prophylactic agents to prevent C. difficile infections in humans. Since these agents do not detectably alter the indigenous gut microbiota or colonization resistance in mice, we believe they will be safe to administer as a prophylactic to block transmission in high-risk environments without rendering patients susceptible to enteric infection after cessation of treatment.
Listeria monocytogenes is a significant foodborne human pathogen that can cause severe disease in certain high-risk individuals. L. monocytogenes is known to produce high-molecular-weight, phage ...tail-like bacteriocins, or "monocins," upon induction of the SOS system. In this work, we purified and characterized monocins and found them to be a new class of F-type bacteriocins. The L. monocytogenes monocin genetic locus was cloned and expressed in Bacillus subtilis, producing specifically targeted bactericidal particles. The receptor binding protein, which determines target cell specificity, was identified and engineered to change the bactericidal spectrum. Unlike the F-type pyocins of Pseudomonas aeruginosa, which are related to lambda-like phage tails, monocins are more closely related to TP901-1-like phage tails, structures not previously known to function as bacteriocins. Monocins therefore represent a new class of phage tail-like bacteriocins. It appears that multiple classes of phage tails and their related bacteriocins have coevolved separately in parallel.
Phage tail-like bacteriocins (PTLBs) are structures widespread among the members of the bacterial kingdom that are evolutionarily related to the DNA delivery organelles of phages (tails). We identified and characterized "monocins" of Listeria monocytogenes and showed that they are related to the tail structures of TP901-1-like phages, structures not previously known to function as bacteriocins. Our results show that multiple types of envelope-penetrating machines have coevolved in parallel to function either for DNA delivery (phages) or as membrane-disrupting bacteriocins. While it has commonly been assumed that these structures were coopted from phages, we cannot rule out the opposite possibility, that ancient phages coopted complex bacteriocins from the cell, which then underwent adaptations to become efficient at translocating DNA.
There is a medical need for antibacterial agents that do not damage the resident gut microbiota or promote the spread of antibiotic resistance. We recently described a prototypic precision ...bactericidal agent, Av-CD291.2, which selectively kills specific
strains and prevents them from colonizing mice. We have since selected two Av-CD291.2-resistant mutants that have a surface (S)-layer-null phenotype due to distinct point mutations in the
gene. Using newly identified bacteriophage receptor binding proteins for targeting, we constructed a panel of Avidocin-CDs that kills diverse
isolates in an S-layer sequence-dependent manner. In addition to bacteriophage receptor recognition, characterization of the mutants also uncovered important roles for S-layer protein A (SlpA) in sporulation, resistance to innate immunity effectors, and toxin production. Surprisingly, S-layer-null mutants were found to persist in the hamster gut despite a complete attenuation of virulence. These findings suggest antimicrobials targeting virulence factors dispensable for fitness in the host force pathogens to trade virulence for viability and would have clear clinical advantages should resistance emerge. Given their exquisite specificity for the pathogen, Avidocin-CDs have substantial therapeutic potential for the treatment and prevention of
infections.
Clostridium difficile causes one of the leading nosocomial infections in developed countries, and therapeutic choices are limited. Some strains of C. difficile produce phage tail-like particles upon ...induction of the SOS response. These particles have bactericidal activity against other C. difficile strains and can therefore be classified as bacteriocins, similar to the R-type pyocins of Pseudomonas aeruginosa. These R-type bacteriocin particles, which have been purified from different strains, each have a different C. difficile-killing spectrum, with no one bacteriocin killing all C. difficile isolates tested. We have identified the genetic locus of these “diffocins” (open reading frames 1359 to 1376) and have found them to be common among the species. The entire diffocin genetic locus of more than 20 kb was cloned and expressed in Bacillus subtilis, and this resulted in production of bactericidal particles. One of the interesting features of these particles is a very large structural protein of ∼200 kDa, the product of gene 1374. This large protein determines the killing spectrum of the particles and is likely the receptor-binding protein. Diffocins may provide an alternate bactericidal agent to prevent or treat infections and to decolonize individuals who are asymptomatic carriers.
Host-adapted strains of Salmonella enterica cause systemic infections and have the ability to persist systemically for long periods of time despite the presence of a robust immune response. ...Chronically infected hosts are asymptomatic and transmit disease to naïve hosts via fecal shedding of bacteria, thereby serving as a critical reservoir for disease. We show that the bacterial effector protein SseI (also called SrfH), which is translocated into host cells by the Salmonella Pathogenicity Island 2 (SPI2) type III secretion system (T3SS), is required for Salmonella typhimurium to maintain a long-term chronic systemic infection in mice. SseI inhibits normal cell migration of primary macrophages and dendritic cells (DC) in vitro, and such inhibition requires the host factor IQ motif containing GTPase activating protein 1 (IQGAP1), an important regulator of cell migration. SseI binds directly to IQGAP1 and co-localizes with this factor at the cell periphery. The C-terminal domain of SseI is similar to PMT/ToxA, a bacterial toxin that contains a cysteine residue (C1165) that is critical for activity. Mutation of the corresponding residue in SseI (C178A) eliminates SseI function in vitro and in vivo, but not binding to IQGAP1. In addition, infection with wild-type (WT) S. typhimurium suppressed DC migration to the spleen in vivo in an SseI-dependent manner. Correspondingly, examination of spleens from mice infected with WT S. typhimurium revealed fewer DC and CD4(+) T lymphocytes compared to mice infected with Delta sseI S. typhimurium. Taken together, our results demonstrate that SseI inhibits normal host cell migration, which ultimately counteracts the ability of the host to clear systemic bacteria.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
A microarray-based negative selection screen was performed to identify Salmonella enterica serovar Typhimurium (serovar Typhimurium) genes that contribute to long-term systemic infection in 129X1/SvJ ...(Nramp1(r)) mice. A high-complexity transposon-mutagenized library was used to infect mice intraperitoneally, and the selective disappearance of mutants was monitored after 7, 14, 21, and 28 d postinfection. One hundred and eighteen genes were identified to contribute to serovar Typhimurium infection of the spleens of mice by 28 d postinfection. The negatively selected mutants represent many known aspects of Salmonella physiology and pathogenesis, although the majority of the identified genes are of putative or unknown function. Approximately 30% of the negatively selected genes correspond to horizontally acquired regions such as those within Salmonella pathogenicity islands (SPI 1-5), prophages (Gifsy-1 and -2 and remnant), and the pSLT virulence plasmid. In addition, mutations in genes responsible for outer membrane structure and remodeling, such as LPS- and PhoP-regulated and fimbrial genes, were also selected against. Competitive index experiments demonstrated that the secreted SPI2 effectors SseK2 and SseJ as well as the SPI4 locus are attenuated relative to wild-type bacteria during systemic infection. Interestingly, several SPI1-encoded type III secretion system effectors/translocases are required by serovar Typhimurium to establish and, unexpectedly, to persist systemically, challenging the present description of Salmonella pathogenesis. Moreover, we observed a progressive selection against serovar Typhimurium mutants based upon the duration of the infection, suggesting that different classes of genes may be required at distinct stages of infection. Overall, these data indicate that Salmonella long-term systemic infection in the mouse requires a diverse repertoire of virulence factors. This diversity of genes presumably reflects the fact that bacteria sequentially encounter a variety of host environments and that Salmonella has evolved to respond to these selective forces in a way that permits both the bacteria and the host to survive.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
Next-Generation Sequencing technology has enhanced oncology research by enabling the detection of all cancer related variants into one assay for research and drug discovery programs. The ...Oncomine࣪ Comprehensive Assay v3, a pan-cancer panel, used with the Ion Torrent࣪ Genexus࣪ System allows for formalin-fixed paraffin embedded (FFPE) samples to be examined across 161 unique genes in an automated sample to result workflow in 30hrs. This study demonstrates ≥ 95% Sensitivity and PPV for detecting SNV, Indel, and Copy Number variants of clinical FFPE samples along with fusions. When tested using analytical controls, the Genexus System achieves ≥ 98% Sensitivity for hotspot variants, ≥ 95% for de novo variants, and 100% Sensitivity for fusion variants. The Ion Torrent࣪ Genexus࣪ System is fully automated and consists of two software linked instruments, the Ion Torrent࣪ Genexus࣪ Purification System and the Ion Torrent࣪ Genexus࣪ Integrated Sequencer. For this study, the purification system was used to sequentially extract and quantify DNA & RNA samples from human colon and lung FFPE tumor tissue. The purification instrument provides minimal hands-on-time, ease of use and compatibility with the integrated sequencer. It extracts and quantifies the nucleic acids, records the quantitation values and transfers the nucleic acids to an output plate. The plate is transferred from the purification instrument directly to the sequencer for sample dilution, library preparation, and sequencing using the Oncomine࣪ Comprehensive Assay v3 panel. Variant calling analysis of the DNA & RNA pairs is completed immediately following sequencing. Results show that the variants are detected reproducibly with ≥ 95% Sensitivity and PPV when compared to the Ion GeneStudio࣪ S5 System as an orthogonal method. All variants in the report were correct with p-values ≤ 10-5. Additionally, a subset of the extracted samples was evaluated using Sanger Sequencing to verify hotspot mutations found in samples sequenced on both the Genexus࣪ System and GeneStudio࣪ S5 System. Results show that all expected hotspot variants were detected. In summary, the Ion Torrent࣪ Genexus࣪ System is a reliable and fast turnaround solution for sample-to-variant calling results. When used with the Oncomine࣪ Comprehensive Assay v3 panel, the system provides accurate identification of tumor markers for oncology research.
Citation Format: Iris Casuga, Frances Chan, Milton Huynh, Gregory R. Govoni, Kayla Zochowski, Thilanka Jayaweera, Janice Au-Young. Rapid and accurate variant calling of FFPE samples with the Genexus System abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2944.
Host-adapted strains of Salmonella enterica cause systemic infections and have the ability to persist systemically for long periods of time despite the presence of a robust immune response. ...Chronically infected hosts are asymptomatic and transmit disease to naïve hosts via fecal shedding of bacteria, thereby serving as a critical reservoir for disease. We show that the bacterial effector protein SseI (also called SrfH), which is translocated into host cells by the Salmonella Pathogenicity Island 2 (SPI2) type III secretion system (T3SS), is required for Salmonella typhimurium to maintain a long-term chronic systemic infection in mice. SseI inhibits normal cell migration of primary macrophages and dendritic cells (DC) in vitro, and such inhibition requires the host factor IQ motif containing GTPase activating protein 1 (IQGAP1), an important regulator of cell migration. SseI binds directly to IQGAP1 and co-localizes with this factor at the cell periphery. The C-terminal domain of SseI is similar to PMT/ToxA, a bacterial toxin that contains a cysteine residue (C1165) that is critical for activity. Mutation of the corresponding residue in SseI (C178A) eliminates SseI function in vitro and in vivo, but not binding to IQGAP1. In addition, infection with wild-type (WT) S. typhimurium suppressed DC migration to the spleen in vivo in an SseI-dependent manner. Correspondingly, examination of spleens from mice infected with WT S. typhimurium revealed fewer DC and CD4+ T lymphocytes compared to mice infected with δsseI S. typhimurium. Taken together, our results demonstrate that SseI inhibits normal host cell migration, which ultimately counteracts the ability of the host to clear systemic bacteria.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Aims. Narrow-angle tailed (NAT) sources in clusters of galaxies can show on the large scale very narrow tails that are unresolved even at arcsecond resolution. These sources could therefore be ...classified as one-sided jets. The aim of this paper is to gain new insight into the structure of these sources, and establish whether they are genuine one-sided objects, or if they are two-sided sources. Methods. We observed a sample of apparently one-sided NAT sources at subarcsecond resolution to obtain detailed information on their structure in the nuclear regions of radio galaxies. Results. Most radio galaxies are found to show two-sided jets with sharp bends, and therefore the sources are similar to the more classical NATs, which are affected by strong projection effects.
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