Primary Duck Hepatocyte Culture and Duck Hepatitis B Virus Infection Model.

Duck hepatitis B virus (DHBV) is an avian member of the hepatotropic DNA viruses, or hepadnaviridae. It shares with the human hepatitis B virus (HBV) a similar genomic organization and replication strategy via reverse transcription, but is simpler than HBV in lacking the X gene and in expressing just two coterminal envelope proteins: Large (L) and small (S). DHBV has been extensively used as a convenient and valuable animal model for study of the hepadnaviral life cycle, and for drug screening in vitro but also in vivo. Ducks and primary duck hepatocytes (PDHs) are inexpensive, easily accessible, and readily infected with DHBV. The high levels of genome replication and protein expression in duck liver and PDHs also facilitate monitoring of viral life cycle using conventional molecular biology techniques such as Southern blot for replicative DNA and covalently closed circular DNA (cccDNA), Northern blot for viral RNAs, and Western blot for viral proteins.

Vaccination with a Human Papillomavirus L2 Multimer Provides Broad Protection against 17 Human Papillomavirus Types in the Mouse Cervicovaginal Challenge Model.

Human papillomavirus (HPV) is a prevalent cause of mucosal and cutaneous infections and underlying conditions ranging from benign warts to anogenital and oropharyngeal cancers affecting both males and females, notably cervical cancer. Cervical cancer is the fourth leading cause of cancer deaths among women globally and is the most impactful in low- and middle-income countries (LMICs), where the costs of screening and licensed L1-based HPV vaccines pose significant barriers to comprehensive administration. Additionally, the licensed L1-based HPV vaccines fail to protect against all oncogenic HPV types. This study generated three independent lots of an L2-based target antigen (LBTA), which was engineered from conserved linear L2-protective epitopes (aa11-88) from five human alphapapillomavirus genotypes in E. coli under cGMP conditions and adjuvanted with aluminum phosphate. Vaccination of rabbits with LBTA generated high neutralizing antibody titers against all 17 HPV types tested, surpassing the nine types covered by Gardasil®9. Passive transfer of naïve mice with LBTA antiserum revealed its capacity to confer protection against vaginal challenge with all 17 αHPV types tested. LBTA shows stability at room temperature over >1 month. Standard in vitro and in vivo toxicology studies suggest a promising safety profile. These findings suggest LBTA's promise as a next-generation vaccine with comprehensive coverage aimed at reducing the economic and healthcare burden of cervical and other HPV+ cancers in LMICs, and it has received regulatory approval for a first-in-human clinical study (NCT05672966).

Transcriptome analysis of CpGV in midguts of type II resistant codling moth larvae and identification of contaminant infections by SNP mapping of RNA-Seq data.

Various isolates of the Cydia pomonella granulovirus (CpGV) are used as insect pest control agents against codling moth (CM, Cydia pomonella L.), a predominant pest in apple orchards. Three different types (I-III) of dominantly inherited field resistance of CM larvae to CpGV have been recently identified. In this study, transcription of virus genes in midgut cells of type II-resistant CM larvae infected with different CpGV isolates, i.e., CpGV-M and CpGV-S (both prone to type II resistance) as well as CpGV-E2 (breaking type II resistance) was determined by strand-specific RNA sequencing (RNA-Seq) at an early infection stage (72 h post infection). Based on principal component analysis of read counts and the quantitative distribution of single nucleotide polymorphisms (SNPs) in the RNA-Seq data, a bioinformatics analysis pipeline was developed for an a posteriori identification of the infective agents. We report that (i) identification of infective agent is crucial, especially in in vivo infection experiments, when activation of covert virus infections is a possibility, (ii) no substantial difference between CpGV-M and CpGV-S transcription was found in type II-resistant CM larvae despite a different resistance mechanism, (iii) the transcription level of CpGV-M and CpGV-S was much lower than that of CpGV-E2, and (iv) orf59 (sod), orf89 (pif-6), orf92 (p18), and orf137 (lef-10) were identified as significantly downregulated genes in resistance-prone isolates CpGV-M and CpGV-S. For type II resistance of CM larvae, we conclude that CpGV-M and CpGV-S are both able to enter midgut cells, but viral transcription is significantly impaired at an early stage of infection compared to the resistance-breaking isolate CpGV-E2. IMPORTANCE: CpGV is a highly virulent pathogen of codling moth, and it has been developed into one of the most successful commercial baculovirus biocontrol agents for pome fruit production worldwide. The emergence of field resistance in codling moth to commercial CpGV products is a threat toward the sustainable use of CpGV. In recent years, different types of resistance (type I-III) were identified. For type II resistance, very little is known regarding the infection process. By studying the virus gene expression patterns of different CpGV isolates in midguts of type II-resistant codling moth larvae, we found that the type II resistance mechanism is most likely based on intracellular factors rather than a receptor component. By applying SNP mapping of the RNA-Seq data, we further emphasize the importance of identifying the infective agents in in vivo experiments when activation of a covert infection cannot be excluded.

Major HBV splice variant encoding a novel protein important for infection.

BACKGROUND & AIMS: HBV expresses more than 10 spliced RNAs from the viral pregenomic RNA, but their functions remain elusive and controversial. To address the function of HBV spliced RNAs, we generated splicing-deficient HBV mutants and conducted experiments to assess the impact of these mutants on HBV infection. METHODS: HepG2-NTCP cells, human hepatocyte chimeric FRG mice (hu-FRG mice), and serum from patients with chronic hepatitis B were used for experiments on HBV infection. Additionally, SHifter assays and cryo-electron microscopy were performed. RESULTS: We found the infectivity of splicing-deficient HBV was decreased 100-1,000-fold compared with that of wild-type HBV in hu-FRG mice. Another mutant, A487C, which loses the most abundant spliced RNA (SP1), also exhibits severely impaired infectivity. SP1 hypothetically encodes a novel protein HBcSP1 (HBc-Cys) that lacks the C-terminal cysteine from full-length HBc. In the SHifter assay, HBcSP1 was detected in wild-type viral particles at a ratio of about 20-100% vs. conventional HBc, as well as in the serum of patients with chronic hepatitis B, but not in A487C particles. When infection was conducted with a shorter incubation time of 4-8 h at lower PEG concentrations in HepG2-NTCP cells, the entry of the A487C mutant was significantly slower. SP1 cDNA complementation of the A487C mutant succeeded in rescuing its infectivity in hu-FRG mice and HepG2-NTCP cells. Moreover, cryo-electron microscopy revealed a disulfide bond between HBc cysteine 183 and 48 in the HBc intradimer of the A487C capsid, leading to a locked conformation that disfavored viral entry in contrast to the wild-type capsid. CONCLUSIONS: Prior studies unveiled the potential integration of the HBc-Cys protein into the HBV capsid. We confirmed the proposal and validated its identity and function during infection. IMPACT AND IMPLICATIONS: HBV SP1 RNA encodes a novel HBc protein (HBcSP1) that lacks the C-terminal cysteine from conventional HBc (HBc-Cys). HBcSP1 was detected in cell culture-derived HBV and confirmed in patients with chronic infection by both immunological and chemical modification assays at 10-50% of capsid. The splicing-deficient mutant HBV (A487C) impaired infectivity in human hepatocyte chimeric mice and viral entry in the HepG2-NTCP cell line. Furthermore, these deficiencies of the splicing-deficient mutant could be rescued by complementation with the SP1-encoded protein HBcSP1. We confirmed and validated the identity and function of HBcSP1 during infection, building on the current model of HBV particles.

Lopinavir and ritonavir act synergistically with azoles against Candida auris in vitro and in a mouse model of disseminated candidiasis.

INTRODUCTION AND OBJECTIVES: The emergence of Candida auris has created a global health challenge. Azole antifungals are the most affected antifungal class because of the extraordinary capability of C. auris to develop resistance against these drugs. Here, we used a combinatorial therapeutic approach to sensitize C. auris to azole antifungals. METHODS AND RESULTS: We have demonstrated the capability of the HIV protease inhibitors lopinavir and ritonavir, at clinically relevant concentrations, to be used with azole antifungals to treat C. auris infections both in vitro and in vivo. Both lopinavir and ritonavir exhibited potent synergistic interactions with the azole antifungals, particularly with itraconazole against 24/24 (100%) and 31/34 (91%) of tested C. auris isolates, respectively. Furthermore, ritonavir significantly interfered with the fungal efflux pump, resulting in a significant increase in Nile red fluorescence by 44%. In a mouse model of C. auris systemic infection, ritonavir boosted the activity of lopinavir to work synergistically with fluconazole and itraconazole and significantly reduced the kidney fungal burden by a 1.2 log (∼94%) and 1.6 log (∼97%) CFU, respectively. CONCLUSION: Our results urge further comprehensive assessment of azoles and HIV protease inhibitors as a novel drug regimen for the treatment of serious invasive C. auris infections.

Overexpression of Leishmania major protein arginine methyltransferase 6 reduces parasite infectivity in vivo.

Arginine methylation is catalysed by Protein Arginine Methyltransferases (PRMTs) and can affect how a target protein functions and how it interacts with other macromolecules, which in turn impacts on cell metabolism and gene expression control. Leishmania parasites express five different PRMTs, and although the presence of each individual PRMT is not essential per se, the imbalanced activity of these PRMTs can impact the virulence of Leishmania parasites in vitro and in vivo. Here we created a Leishmania major cell line overexpressing PRMT6 and show that similar to what was observed for the T. brucei homologous enzyme, L. major PRMT6 probably has a narrow substrate range. However, its overexpression notably impairs the infection in mice, with a mild reduction in the number of viable parasites in the lymph nodes. Our results indicate that arginine methylation by LmjPRMT6 plays a significant role in the adaptation of the parasite to the environment found in the mammalian host.

Brazilian Red Propolis Presents Promising Anti-H. pylori Activity in In Vitro and In Vivo Assays with the Ability to Modulate the Immune Response.

Helicobacter pylori is a Gram-negative, microaerophilic, curved-rod, flagellated bacterium commonly found in the stomach mucosa and associated with different gastrointestinal diseases. With high levels of prevalence worldwide, it has developed resistance to the antibiotics used in its therapy. Brazilian red propolis has been studied due to its biological properties, and in the literature, it has shown promising antibacterial activities. The aim of this study was to evaluate anti-H. pylori from the crude hydroalcoholic extract of Brazilian red propolis (CHEBRP). For this, in vitro determination of the minimum inhibitory and bactericidal concentration (MIC/MBC) and synergistic activity and in vivo, microbiological, and histopathological analyses using Wistar rats were carried out using CHEBRP against H. pylori strains (ATCC 46523 and clinical isolate). CHEBRP presented MIC/MBC of 50 and 100 µg/mL against H. pylori strains (ATCC 43526 and clinical isolate, respectively) and tetracycline MIC/MBC of 0.74 µg/mL. The association of CHEBRP with tetracycline had an indifferent effect. In the stomach mucosa of rats, all treatments performed significantly decreased the number of H. pylori, and a concentration of 300 mg/kg was able to modulate the inflammatory response in the tissue. Therefore, CHEBRP showed promising anti-H. pylori in in vitro and in vivo assays.

Using Genomic Deletion Mutants to Investigate Effector-Triggered Immunity During Legionella pneumophila Infection.

Legionella pneumophila is an intracellular bacterial pathogen that uses a type IV secretion system (T4SS), termed Dot/Icm, to secrete more than 330 virulence effector proteins into the infected host cell. Many Dot/Icm effectors are involved in biogenesis of the Legionella-containing vacuole (LCV), which allows intracellular bacterial replication in environmental amoebae and alveolar macrophages. Through their activity, some effectors trigger the mammalian host immune response in a phenomenon termed effector-triggered immunity (ETI). Here, we describe a protocol to create and use L. pneumophila genome deletion mutants to identify effector(s) that alter pro-inflammatory cytokine production and bacterial clearance in the lungs of mice.

Design and Characterization of Myristoylated and Non-Myristoylated Peptides Effective against Candida spp. Clinical Isolates.

The increasing resistance of fungi to antibiotics is a severe challenge in public health, and newly effective drugs are required. Promising potential medications are lipopeptides, linear antimicrobial peptides (AMPs) conjugated to a lipid tail, usually at the N-terminus. In this paper, we investigated the in vitro and in vivo antifungal activity of three short myristoylated and non-myristoylated peptides derived from a mutant of the AMP Chionodracine. We determined their interaction with anionic and zwitterionic membrane-mimicking vesicles and their structure during this interaction. We then investigated their cytotoxic and hemolytic activity against mammalian cells. Lipidated peptides showed a broad spectrum of activity against a relevant panel of pathogen fungi belonging to Candida spp., including the multidrug-resistant C. auris. The antifungal activity was also observed vs. biofilms of C. albicans, C. tropicalis, and C. auris. Finally, a pilot efficacy study was conducted on the in vivo model consisting of Galleria mellonella larvae. Treatment with the most-promising myristoylated peptide was effective in counteracting the infection from C. auris and C. albicans and the death of the larvae. Therefore, this myristoylated peptide is a potential candidate to develop antifungal agents against human fungal pathogens.

Growth-phase specific regulation of cviI/R based quorum sensing associated virulence factors in Chromobacterium violaceum by linalool, a monoterpenoid.

Quorum sensing (QS)-dependent gene regulation in bacteria performs a vital role in synchronization of cell-density-dependent functions. In Chromobacterium violaceum QS-dependent cviI/R regulatory genes are activated during the mid- or late-exponential phase of growth. However, sufficient evidence is lacking on the role of QS inhibitors on gene regulation at different phases of growth. Hence, we report the role of linalool, a natural monoterpenoid on QS mediated gene regulation at different stages of growth in C. violaceum by performing biosensor, growth kinetic and gene expression studies. In vitro and in vivo studies were performed for establishing role of linalool in reducing the virulence and infection by using HEK-293 T cell lines and Caenorhabditis elegans models respectively. C. violaceum CV026 with C6-HSL was used as control. The results showed linalool to be a QS inhibitor with an estimated IC50 of 63 µg/mL for violacein inhibition. At this concentration the cell density difference (delta OD600) of 0.14 from the compound was observed indicating the quorum concentration. The expression of cviI/R was initiated at mid-log phase (~ 18 h) and reached the maximum at 36 h in control whereas in treatment it remained significantly downregulated at all time points. The expression of violacein biosynthetic genes vioA, vioC, vioD and vioE was also downregulated by linalool. Infection studies with linalool showed higher survival rates in HEK-293T cell lines and C. elegans compared to the infection control. Taken together, this study proves linalool to be a QS inhibitor capable of attenuation of QS by controlling the cell density through cviI/R downregulation at the early phase of growth and hence offering scope for its application for controlling infections.

IκBNS-deficiency protects mice from fatal Listeria monocytogenes infection by blunting pro-inflammatory signature in Ly6Chigh monocytes and preventing exaggerated innate immune responses.

IκB proteins regulate the inhibition and activation of NF-κB transcription factor complexes. While classical IκB proteins keep NF-κB complexes inactive in the cytoplasm, atypical IκB proteins act on activated NF-κB complexes located in the nucleus. Most of the knowledge regarding the function of IκB proteins has been collected in vitro, while far less is known regarding their impact on activation and regulation of immune responses during in vivo infections. Combining in vivo Listeria monocytogenes (Lm) infection with comparative ex vivo transcriptional profiling of the hepatic response to the pathogen we observed that in contrast to wild type mice that mounted a robust inflammatory response, IκBNS-deficiency was generally associated with a transcriptional repression of innate immune responses. Whole tissue transcriptomics revealed a pronounced IκBNS-dependent reduction of myeloid cell-associated transcripts in the liver together with an exceptionally high Nfkbid promoter activity uncovered in Ly6Chigh inflammatory monocytes prompted us to further characterize the specific contribution of IκBNS in the inflammatory response of monocytes to the infectious agent. Indeed, Ly6Chigh monocytes primed during Lm infection in the absence of IκBNS displayed a blunted response compared to wild type-derived Ly6Chigh monocytes as evidenced by the reduced early expression of hallmark transcripts of monocyte-driven inflammation such as Il6, Nos2 and Il1ß. Strikingly, altered monocyte activation in IκBNS-deficient mice was associated with an exceptional resistance against Lm infection and protection was associated with a strong reduction in immunopathology in Lm target organs. Of note, mice lacking IκBNS exclusively in myeloid cells failed to resist Lm infection, indicating that the observed effect was not monocyte intrinsic but monocyte extrinsic. While serum cytokine-profiling did not discover obvious differences between wild type and IκBNS -/- mice for most of the analyzed mediators, IL-10 was virtually undetectable in IκBNS-deficient mice, both in the steady state and following Lm infection. Together, we show here a crucial role for IκBNS during Lm infection with IκBNS-deficient mice showing an overall blunted pro-inflammatory immune response attributed to a reduced pro-inflammatory signature in Ly6Chigh monocytes. Reduced immunopathology and complete protection of mice against an otherwise fatal Lm infection identified IκBNS as molecular driver of inflammation in listeriosis.

A non-classical monocyte-derived macrophage subset provides a splenic replication niche for intracellular Salmonella.

Interactions between intracellular bacteria and mononuclear phagocytes give rise to diverse cellular phenotypes that may determine the outcome of infection. Recent advances in single-cell RNA sequencing (scRNA-seq) have identified multiple subsets within the mononuclear population, but implications to their function during infection are limited. Here, we surveyed the mononuclear niche of intracellular Salmonella Typhimurium (S.Tm) during early systemic infection in mice. We described eclipse-like growth kinetics in the spleen, with a first phase of bacterial control mediated by tissue-resident red-pulp macrophages. A second phase involved extensive bacterial replication within a macrophage population characterized by CD9 expression. We demonstrated that CD9+ macrophages induced pathways for detoxificating oxidized lipids, that may be utilized by intracellular S.Tm. We established that CD9+ macrophages originated from non-classical monocytes (NCM), and NCM-depleted mice were more resistant to S.Tm infection. Our study defines macrophage subset-specific host-pathogen interactions that determine early infection dynamics and infection outcome of the entire organism.

Targeted RNA-Seq Reveals the M. tuberculosis Transcriptome from an In Vivo Infection Model.

The study of host-pathogen interactions using in vivo models with intracellular pathogens like Mycobacterium tuberculosis (Mtb) entails technical limitations, such as: (i) Selecting an efficient differential lysis system to enrich the pathogen cells; (ii) obtaining sufficient high-quality RNA; and (iii) achieving an efficient rRNA depletion. Thus, some authors had used flow cytometers to separate infected cells or significantly increase the sequencing depth of host-pathogen RNA libraries to observe the pathogens' gene expression. However, these options carry additional expenses in specialized equipment typically not available for all laboratories. Here, we propose an experimental protocol involving differential cell lysis and a probe-based ribosomal depletion to determine the gene expression of Mtb and its host during in vivo infection. This method increased the number of observed pathogen-expressed genes from 13 using the traditional RNA-seq approach to 702. After eliminating rRNA reads, we observed that 61.59% of Mtb sequences represented 702 genes, while 38.41% represented intergenic regions. Some of the most expressed genes codified for IS1081 (Rv2512c) transposase and eight PE-PGRS members, such as PGRS49 and PGRS50. As expected, a critical percent of the expressed genes codified for secreted proteins essential for infection, such as PE68, lppN, and LpqH. Moreover, three Mtb ncRNAs were highly expressed (small RNA MTS2823, transfer-messenger RNA RF00023, and ribozyme RF00010). Many of the host-expressed genes were related to the inflammation process and the expression of surfactant proteins such as the Sftpa and Sftpc, known to bind Mtb to alveolar macrophages and mi638, a microRNA with no previous associations with pulmonary diseases. The main objective of this study is to present the method, and a general catalog of the Mtb expressed genes at one point of the in vivo infection. We believe our method represents a different approach to the existing ones to study host-pathogen interactions in tuberculosis and other similar intracellular infections, without the necessity of specialized equipment.

Type VI Secretion System and Its Effectors PdpC, PdpD, and OpiA Contribute to Francisella Virulence in Galleria mellonella Larvae.

Francisella tularensis causes the deadly zoonotic disease tularemia in humans and is able to infect a broad range of organisms including arthropods, which are thought to play a major role in Francisella transmission. However, while mammalian in vitro and in vivo infection models are widely used to investigate Francisella pathogenicity, a detailed characterization of the major Francisella virulence factor, a noncanonical type VI secretion system (T6SS), in an arthropod in vivo infection model is missing. Here, we use Galleria mellonella larvae to analyze the role of the Francisella T6SS and its corresponding effectors in F. tularensis subsp. novicida virulence. We report that G. mellonella larvae killing depends on the functional T6SS and infectious dose. In contrast to other mammalian in vivo infection models, even one of the T6SS effectors PdpC, PdpD, or OpiA is sufficient to kill G. mellonella larvae, while sheath recycling by ClpB is dispensable. We further demonstrate that treatment by polyethylene glycol (PEG) activates Francisella T6SS in liquid culture and that this is independent of the response regulator PmrA. PEG-activated IglC secretion is dependent on T6SS structural component PdpB but independent of putative effectors PdpC, PdpD, AnmK, OpiB1, OpiB2, and OpiB3. The results of larvae infection and secretion assay suggest that AnmK, a putative T6SS component with unknown function, interferes with OpiA-mediated toxicity but not with general T6SS activity. We establish that the easy-to-use G. mellonella larvae infection model provides new insights into the function of T6SS and pathogenesis of Francisella.

Gene expression patterns of Cydia pomonella granulovirus in codling moth larvae revealed by RNAseq analysis.

The Cydia pomonella granulovirus (CpGV) has been used as a biological control agent of codling moth (Cydia pomonella), a severe global pest on pome fruit. Despite the economic importance, our knowledge of its molecular biology is still limited and a detailed picture of its gene expression is still missing. Here, we sequenced the transcriptome of codling moth larvae infected with the Mexican isolate CpGV-M and analyzed the expression of viral genes at 12, 48, and 96 h post infection (hpi). The results showed that two genes (p6.9 and pp31/39K) related to DNA binding of virus production, were highly expressed at 48 and 96 hpi. From 48 to 96 hpi, the expression of genes associated with virus replication and dissemination decreased, whereas the expression of genes related to infectious virion production and per os infectivity increased. This study provides a comprehensive view of CpGV gene expression patterns in host larvae.

In vivo infection of Bursaphelenchus xylophilus by the fungus Esteya vermicola.

BACKGROUND: As the causal agent of pine wilt disease, Bursaphelenchus xylophilus, is a serious pathogen of forest pine trees. Esteya vermicola is a nematophagous fungus of B. xylophilus and exhibits great potential as a biological control agent. However, the in vivo infection mechanism of E. vermicola on B. xylophilus is unclear. Experiments were conducted to study the colonization of host plant and infection of B. xylophilus by E. vermicola inside pine tree xylem. RESULTS: A green fluorescent protein (GFP)-tagged E. vermicola transformant was constructed as a biomarker to study the in vivo colonization and infection of B. xylophilus in pine trees. The in vitro infection of B. xylophilus by E. vermicola was observed through GFP expression. The bacilloid conidia produced by trophic hyphae in the body of the nematode are described. Additionally, the monitoring of in vivo colonization by GFP-tagged E. vermicola showed the germination and hyphal extension of this fungus after inoculation. Moreover, B. xylophilus infected by this biocontrol agent were extracted from healthy seedlings and observed in the xylem of trees that were wilting due to pine wilt disease. CONCLUSION: Evidence of fungal colonization and infection of B. xylophilus by E. vermicola is provided to improve our understanding of the in vivo infection mechanisms used by this nematophagous fungus against B. xylophilus. The infection of B. xylophilus by E. vermicola was inferred to begin with the implantation of propagules, and this inference will require future investigation. The colonization of Esteya vermicola in host pine tree xylem and the in vivo infection of pinewood nematode by E. vermicola were investigated using the green fluorescence protein transformant. © 2020 Society of Chemical Industry.

Efficacy of newly generated short antimicrobial cationic lipopeptides against methicillin-resistant Staphylococcus aureus (MRSA).

INTRODUCTION: Infection caused by methicillin-resistant Staphylococcus aureus (S. aureus) (MRSA) is a serious clinical challenge and research to develop new antimicrobials is imperative. METHODS: This study investigated the in vitro and in vivo efficacy of the short cationic dialkyl lipopeptides (C10)2-KKKK-NH2 and (C12)2-KKKK-NH2. The antibacterial efficacy of (C10)2-KKKK-NH2 and (C12)2-KKKK-NH2 was evaluated in representative clinical methicillin-susceptible S. aureus and MRSA strains by both in vitro (MIC, time-kill curve) and in vivo (wax worms model) approaches. RESULTS: These studies revealed that both (C10)2-KKKK-NH2 and (C12)2-KKKK-NH2 have rapid bactericidal activity, with a decrease of > 3 log10 colony forming units (CFU)/mL achieved in the first 6 hours of treatment. Furthermore, (C10)2-KKKK-NH2 performed similarly to daptomycin, with a sustained bacterial killing after 24 hours. Wax worms infected and treated with these lipopeptides showed a decreased survival rate of 90% to 50% within the first day of treatment. Scanning electron microscopy determined that the effect of the short lipopeptides in S. aureus was associated with important morphological structural changes that may suggest cell membrane perturbation. CONCLUSION: These findings suggest that the short lipopeptides (C10)2-KKKK-NH2 and (C12)2-KKKK-NH2 may be potential new options for treating MRSA infections.

Mapping of Shigella flexneri's tissue distribution and type III secretion apparatus activity during infection of the large intestine of guinea pigs.

Shigella spp. are bacterial pathogens that invade the human colonic mucosa using a type III secretion apparatus (T3SA), a proteinaceous device activated upon contact with host cells. Active T3SAs translocate proteins that carve the intracellular niche of Shigella spp. Nevertheless, the activation state of the T3SA has not been addressed in vivo. Here, we used a green fluorescent protein transcription-based secretion activity reporter (TSAR) to provide a spatio-temporal description of S. flexneri T3SAs activity in the colon of Guinea pigs. First, we observed that early mucus release is triggered in the vicinity of luminal bacteria with inactive T3SA. Subsequent mucosal invasion showed bacteria with active T3SA associated with the brush border, eventually penetrating into epithelial cells. From 2 to 8 h post-challenge, the infection foci expanded, and these intracellular bacteria displayed homogeneously high-secreting activity, while extracellular foci within the lamina propria featured bacteria with low secretion activity. We also found evidence that within lamina propria macrophages, bacteria reside in vacuoles instead of accessing the cytosol. Finally, bacteria were cleared from tissues between 8 and 24 h post-challenge, highlighting the hit-and-run colonization strategy of Shigella. This study demonstrates how genetically encoded reporters can contribute to deciphering pathogenesis in vivo.

Essential role of IκBNS for in vivo CD4+ T-cell activation, proliferation, and Th1-cell differentiation during Listeria monocytogenes infection in mice.

Acquisition of effector functions in T cells is guided by transcription factors, including NF-κB, that itself is tightly controlled by inhibitory proteins. The atypical NF-κB inhibitor, IκBNS, is involved in the development of Th1, Th17, and regulatory T (Treg) cells. However, it remained unclear to which extend IκBNS contributed to the acquisition of effector function in T cells specifically responding to a pathogen during in vivo infection. Tracking of adoptively transferred T cells in Listeria monocytogenes infected mice antigen-specific activation of CD4+ T cells following in vivo pathogen encounter to strongly rely on IκBNS . While IκBNS was largely dispensable for the acquisition of cytotoxic effector function in CD8+ T cells, IκBNS -deficient Th1 effector cells exhibited significantly reduced proliferation, marked changes in the pattern of activation marker expression, and reduced production of the Th1-cell cytokines IFN-γ, IL-2, and TNF-α. Complementary in vitro analyses using cells from novel reporter and inducible knockout mice revealed that IκBNS predominantly affects the early phase of Th1-cell differentiation while its function in terminally differentiated cells appears to be negligible. Our data suggest IκBNS as a potential target to modulate specifically CD4+ T-cell responses.

Responses of marine mussel Mytilus galloprovincialis (Bivalvia: Mytilidae) after infection with the pathogen Vibrio splendidus.

Bivalve molluscs possess effective cellular and humoral defence mechanisms against bacterial infection. Although the immune responses of mussels to challenge with pathogenic vibrios have been largely investigated, the effects at the site of injection at the tissue level have not been so far evaluated. To this aim, mussels Mytilus galloprovincialis were herein in vivo challenged with Vibrio splendidus to assess the responses induced in hemolymph and posterior adductor muscle (PAM), being the site of bacterial infection. The number of living intra-hemocyte bacteria increased after the first hour post-injection (p.i.), suggesting the occurrence of an intense phagocytosis, while clearance was observed within 24 h p.i. A recruitment of hemocytes at the injection site was found in mussel PAM, together with marked morphological changes in the volume of muscular fibers, with a recovery of muscle tissue organization after 48 h p.i. A concomitant impairment in the osmoregulatory processes were observed in PAM by an initial inhibition of aquaporins and increased immunopositivity of Na+/K+ ATPase ionic pump, strictly related to the histological alterations and hemocyte infiltration detected in PAM. Accordingly, an intense cell turnover activity was also recorded following the infection event. Overall, results indicated the hemolymph as the system responsible for the physiological adaptations in mussels to stressful factors, such as pathogenicity, for the maintenance of homeostasis and immune defence. Also, the osmotic balance and cell turnover can be used as objective diagnostic criteria to evaluate the physiological state of mussels following bacterial infection, which may be relevant in aquaculture and biomonitoring studies.