Bacterial biofilms are organized sessile communities of bacteria enclosed in extracellular polymeric substances (EPS). To analyze organization of bacteria and EPS in high resolution and high ...magnification by scanning electron microscopy (SEM), it is important to preserve the complex architecture of biofilms. Therefore, fixation abilities of formalin, glutaraldehyde, and Methacarn (methanol/chloroform/acetic acid-6:3:1) fixatives were evaluated to identify which fixative would best preserve the complex structure of bacterial biofilms. Economically important Gram-negative Mannheimia haemolytica, the major pathogen associated with bovine respiratory disease complex, and Gram-positive Staphylococcus aureus, the major cause of chronic mastitis in cattle, bacteria were selected since both form biofilms on solid-liquid interface. For SEM analysis, round glass coverslips were placed into the wells of 24-well plates and diluted M. haemolytica or S. aureus cultures were added, and incubated at 37°C for 48-72 h under static growth conditions. Culture media were aspirated and biofilms were fixed with an individual fixative for 48 h. SEM examination revealed that all three fixatives were effective preserving the bacterial cell morphology, however only Methacarn fixative could consistently preserve the complex structure of biofilms. EPS layers were clearly visible on the top, in the middle, and in the bottom of the biofilms with Methacarn fixative. Biomass and three-dimensional structure of the biofilms were further confirmed spectrophotometrically following crystal violet staining and by confocal microscopy after viability staining. These findings demonstrate that Methacarn fixative solution is superior to the other fixatives evaluated to preserve the complex architecture of biofilms grown on glass coverslips for SEM evaluation.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Despite decades of focus on crickets (family: Gryllidae) as a popular commodity and model organism, we still know very little about their immune responses to microbial pathogens. Previous studies ...have measured downstream immune effects (e.g., encapsulation response, circulating hemocytes) following an immune challenge in crickets, but almost none have identified and quantified the expression of immune genes during an active pathogenic infection. Furthermore, the prevalence of covert (i.e., asymptomatic) infections within insect populations is becoming increasingly apparent, yet we do not fully understand the mechanisms that maintain low viral loads. In the present study, we measured the expression of several genes across multiple immune pathways in
crickets with an overt or covert infection of cricket iridovirus (CrIV). Crickets with overt infections had higher relative expression of key pathway component genes across the Toll, Imd, Jak/STAT, and RNAi pathways. These results suggests that crickets can tolerate low viral infections but can mount a robust immune response during an overt CrIV infection. Moreover, this study provides insight into the immune strategy of crickets following viral infection and will aid future studies looking to quantify immune investment and improve resistance to pathogens.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
In Escherichia coli O157:H7 (O157), the filamentous structure of the type III secretion system is produced from the polymerization of the EspA protein. EspA filaments are essential for O157 adherence ...to epithelial cells. In previous studies, we demonstrated that O157 hha deletion mutants showed increased adherence to HEp-2 cells and produced abundant biofilms. Transcriptional analysis revealed increased expression of espA as well as the csgA gene, which encodes curli fimbriae that are essential for biofilm formation. In the present study, we constructed hha espA, hha csgA, and hha csgA espA deletion mutants to determine the relative importance of EspA and CsgA in O157 adherence to HEp-2 cells and biofilm formation. In vitro adherence assays, conducted at 37°C in a tissue culture medium containing 0.1% glucose, showed that HEp-2 cell adherence required EspA because hha espA and hha csgA espA mutants adhered to HEp-2 cells at higher levels only when complemented with an espA-expressing plasmid. Biofilm assays performed at 28°C in a medium lacking glucose showed dependency of biofilm formation on CsgA; however EspA was not produced under these conditions. Despite production of detectable levels of EspA at 37°C in media supplemented with 0.1% glucose, the biofilm formation occurred independent of EspA. These results indicate dependency of O157 adherence to epithelial cells on EspA filaments, while CsgA promoted biofilm formation under conditions mimicking those found in the environment (low temperature with nutrient limitations) and in the digestive tract of an host animal (higher temperature and low levels of glucose).
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Super-shed (SS) Escherichia coli O157 (E. coli O157) demonstrate a strong, aggregative, locus of enterocyte effacement (LEE)-independent adherence phenotype on bovine recto-anal junction squamous ...epithelial (RSE) cells, and harbor polymorphisms in non-LEE-adherence-related loci, including in the type 1 fimbriae operon. To elucidate the role of type 1 fimbriae in strain- and host-specific adherence, we evaluated the entire Fim operon (FimB-H) and its adhesion (FimH) deletion mutants in four E. coli O157 strains, SS17, SS52, SS77 and EDL933, and evaluated the adherence phenotype in bovine RSE and human HEp-2 adherence assays. Consistent with the prevailing dogma that fimH expression is genetically switched off in E. coli O157, the ΔfimHSS52, ΔfimB-HSS52, ΔfimB-HSS17, and ΔfimHSS77 mutants remained unchanged in adherence phenotype to RSE cells. In contrast, the ΔfimHSS17 and ΔfimB-HSS77 mutants changed from a wild-type strong and aggregative, to a moderate and diffuse adherence phenotype, while both ΔfimHEDL933 and ΔfimB-HEDL933 mutants demonstrated enhanced binding to RSE cells (p < 0.05). Additionally, both ΔfimHSS17 and ΔfimHEDL933 were non-adherent to HEp-2 cells (p < 0.05). Complementation of the mutant strains with their respective wild-type genes restored parental phenotypes. Microscopy revealed that the SS17 and EDL933 strains indeed carry type 1 fimbriae-like structures shorter than those seen in uropathogenic E. coli. Taken together, these results provide compelling evidence for a strain and host cell type-dependent role of fimH and the fim operon in E. coli O157 adherence that needs to be further evaluated.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
We describe here the gross and microscopic lesions in 18 experimentally induced and 120 natural Campylobacter abortions. In natural Campylobacter abortions, gross lesions were reported infrequently; ...placentitis was recorded in 6% and hepatic lesions in 4% of our field cases. Placentitis was the microscopic lesion identified most consistently in natural abortions (93%) and was often observed in association with abundant bacterial colonies in chorionic villi (54%) and less often with placental vasculitis (13%). In natural abortions, suppurative fetal pneumonia (48%), necrosuppurative hepatitis (16%), and purulent meningitis (7%) were also observed. The better-preserved specimens from experimentally induced abortions were utilized to define placental changes more precisely. Placentitis was identified in all 18 experimentally induced abortions and was observed most consistently in the chorionic villus stroma (100%), often accompanied by suppurative surface exudate (89%). An inflammatory infiltrate was less commonly identified in the cotyledonary hilus (39%) and intercotyledonary placenta (22%). Bacteria were visualized in H&E-stained sections in 89% of placentas from experimentally infected ewes, primarily as well-demarcated bacterial colonies within subtrophoblastic, sinusoidal capillaries (89%), in the cotyledonary villus stroma (89%), and within the cytoplasm of trophoblasts (22%). Transmission electron microscopy and immunohistochemistry confirmed that the vast majority of the well-demarcated bacterial colonies characteristic of Campylobacter abortion were within subtrophoblastic sinusoidal capillaries. The most characteristic microscopic lesions identified in cases of Campylobacter abortion in sheep were placentitis with placental bacterial colonies, placental vasculitis, and fetal pneumonia.
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NUK, OILJ, SAZU, UKNU, UL, UM, UPUK
Peyer’s patches (PPs) are B cell-rich sites of intestinal immune induction, yet PP-associated B cell signaling, activation, and differentiation are poorly defined. Single-cell and spatial ...transcriptomics were completed to study B cells from porcine jejunum and ileum containing PPs. Intestinal locations had distinct immune landscapes, including more follicular B cells in ileum and increased MHC-II-encoding gene expression in jejunal B cells. Despite distinct landscapes, conserved B cell dynamics were detected across intestinal locations, including B cell signaling to CD4+ macrophages that are putative phagocytic, cytotoxic, effector cells and deduced routes of B cell activation/differentiation, including resting B cells migrating into follicles to replicate/divide or differentiate into antibody-secreting cells residing in intestinal crypts. A six-biomarker panel recapitulated transcriptomics findings of B cell phenotypes, frequencies, and spatial locations via ex vivo and in situ staining. Findings convey conserved B cell dynamics across intestinal locations containing PPs, despite location-specific immune environments. Results establish a benchmark of B cell dynamics for understanding intestinal immune induction important to promoting gut/overall health.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Leptospirosis is a worldwide zoonosis caused by pathogenic species of the genus
. The recent application of CRISPR interference (CRISPRi) to
facilitates targeted gene silencing and provides a new ...tool to investigate pathogenic mechanisms of leptospirosis. CRISPRi relies on the expression of a catalytically "dead" Cas9 (dCas9) and a single-guide RNA (sgRNA). Previously, our group generated a LipL32 and a double LigA/LigB (LigAB) mutant, which, in the current study, are characterized by whole-cell proteomics in comparison with control leptospires harboring plasmid expressing dCas9 alone. Comparison of control and LigAB mutant leptospires identified 46 significantly differentially expressed (DE) proteins, including 27 proteins that were less abundant and 19 proteins that were more abundant in the LigAB mutant compared with the control. Comparison of the control and LipL32 mutant leptospires identified 243 DE proteins, of which 84 proteins were more abundant and 159 were less abundant in the LipL32 mutant strain. Significantly increased amounts of known virulence impactors and surface membrane receptors, including LipL45, LipL31, LigB, and LipL41, were identified. The virulence of LipL32 and LigAB mutants were evaluated in the hamster model of leptospirosis; the LigAB mutant was unable to cause acute disease although mutant leptospires could still be recovered from target organs, albeit at a significantly lower bacterial burden (<850 and <16-fold in liver and kidney, respectively, in comparison with control), indicating attenuation of virulence and a shift to chronic bacterial persistence. Notably, the LipL32 mutant displayed augmented virulence as evidenced by early onset of clinical symptoms and increased numbers of circulating foamy macrophages. Validation of LipL32 and LigAB mutants recovered from liver and kidney in the presence or absence of antibiotic selection revealed high plasmid stability and, by extension, gene silencing
. Collectively, this work emphasizes the advantages and feasibility of using CRISPRi technology to evaluate and characterize virulence factors of leptospires and their respective host-pathogen interactions in animal models of leptospirosis. Importantly, it also provides insight into the requirements of LigA and LigB for acute disease and explores the impact of silencing expression of
, which resulted in substantial changes in amounts of outer membrane proteins.
Leptospirosis is a world-wide zoonotic disease caused by pathogenic Leptospira and can be asymptomatic or can cause clinical signs ranging from influenza-like to multi-organ failure and death in ...severe cases. While species and strain specificity can play a major role in disease presentation, the hamster is susceptible to most leptospiral infections and is the model of choice for vaccine efficacy testing. During evaluation of blood smears from hamsters challenged with different species and strains of Leptospira, a circulating population of large, mononuclear, lipid-filled cells, most similar to foamy macrophages (FMs), was detected. Circulating FMs were identified by Giemsa staining and verified by scanning and transmission electron microscopy. FMs were found in the circulating blood of all Leptospira-challenged hamsters, indicating that the finding was not species or strain specific, although higher numbers of FMs tended to correlate with severity of disease. The unique finding of circulating FMs in the hamster model of leptospirosis can yield additional insights into the pathogenesis of leptospirosis and other diseases that induce circulating FMs.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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is a commensal bacterium of the upper respiratory tract in pigs and also the causative agent of Glässer's disease, which causes significant morbidity and mortality in pigs worldwide. Isolates are ...characterized into 15 serovars by their capsular polysaccharide, which has shown a correlation with isolate pathogenicity. To investigate the role the capsule plays in
virulence and host interaction, a capsule mutant of the serovar 5 strain HS069 was generated (HS069Δcap) through allelic exchange following natural transformation. HS069Δcap was unable to cause signs of systemic disease during a pig challenge study and had increased sensitivity to complement killing and phagocytosis by alveolar macrophages. Compared with the parent strain, HS069Δcap produced more robust biofilm and adhered equivalently to 3D4/31 cells; however, it was unable to persistently colonize the nasal cavity of inoculated pigs, with all pigs clearing HS069Δcap by 5 days postchallenge. Our results indicate the importance of the capsular polysaccharide to
virulence as well as nasal colonization in pigs.
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