RESUMO

This chapter introduces protocols for culturing and maintaining Dictyostelium discoideum and methods for conducting virulence assays in this organism to study bacterial pathogenicity. It outlines advanced techniques, such as automated microscopy and flow cytometry, for detailed cellular analysis and traditional microbiological approaches. These comprehensive protocols will enable researchers to probe the virulence factors of pathogens like Klebsiella pneumoniae and to elucidate the details of host-pathogen interactions within a cost-effective and adaptable laboratory framework.

Assuntos

RESUMO

Studying host-pathogen interactions is essential for understanding infectious diseases and developing possible treatments, especially for priority pathogens with increased virulence and antibiotic resistance, such as Klebsiella pneumoniae. Over time, this subject has been approached from different perspectives, often using mammal host models and invasive endpoint measurements (e.g., sacrifice and organ extraction). However, taking advantage of technological advances, it is now possible to follow the infective process by noninvasive visualization in real time, using optically amenable surrogate hosts. In this line, this chapter describes a live-cell imaging approach to monitor the interaction of K. pneumoniae and potentially other bacterial pathogens with zebrafish larvae in vivo. This methodology is based on the microinjection of fluorescent bacteria into the otic vesicle, followed by time-lapse observation by automated fluorescence microscopy with environmental control, monitoring the dynamics of immune cell recruitment, bacterial load, and larvae survival.

Assuntos

RESUMO

The rise of antibiotic-resistant bacterial strains represents an important challenge for global health, underscoring the critical need for innovative strategies to confront this threat. Natural products and their derivatives have emerged as a promising reservoir for drug discovery. The social amoeba Dictyostelium discoideum is a potent model organism in this effort. Employing this invertebrate model, we introduce a novel perspective to investigate natural plant extracts in search of molecules with potential antivirulence activity. Our work established an easy-scalable developmental assay targeting a virulent strain of Klebsiella pneumoniae, with Helenium aromaticum as the representative plant. The main objective was to identify tentative compounds from the Helenium aromaticum extract that attenuate the virulence of K. pneumoniae virulence without inducing cytotoxic effects on amoeba cells. Notably, the methanolic root extract of H. aromaticum fulfilled these prerequisites compared to the dichloromethane extract. Using UHPLC Q/Orbitrap/ESI/MS/MS, 63 compounds were tentatively identified in both extracts, 47 in the methanolic and 29 in the dichloromethane, with 13 compounds in common. This research underscores the potential of employing D. discoideum-assisted pharmacognosy to discover new antivirulence agents against multidrug-resistant pathogens.

RESUMO

BACKGROUND: The convergence of hypervirulence and carbapenem resistance in the bacterial pathogen Klebsiella pneumoniae represents a critical global health concern. Hypervirulent K. pneumoniae (hvKp) strains, frequently from sequence type 23 (ST23) and having a K1 capsule, have been associated with severe community-acquired invasive infections. Although hvKp were initially restricted to Southeast Asia and primarily antibiotic-sensitive, carbapenem-resistant hvKp infections are reported worldwide. Here, within the carbapenemase production Enterobacterales surveillance system headed by the Chilean Public Health Institute, we describe the isolation in Chile of a high-risk ST23 dual-carbapenemase-producing hvKp strain, which carbapenemase genes are encoded in a single conjugative plasmid. RESULTS: Phenotypic and molecular tests of this strain revealed an extensive resistance to at least 15 antibiotic classes and the production of KPC-2 and VIM-1 carbapenemases. Unexpectedly, this isolate lacked hypermucoviscosity, challenging this commonly used hvKp identification criteria. Complete genome sequencing and analysis confirmed the K1 capsular type, the KpVP-1 virulence plasmid, and the GIE492 and ICEKp10 genomic islands carrying virulence factors strongly associated with hvKp. Although this isolate belonged to the globally disseminated hvKp clonal group CG23-I, it is unique, as it formed a clade apart from a previously reported Chilean ST23 hvKp isolate and acquired an IncN KPC-2 plasmid highly disseminated in South America (absent in other hvKp genomes), but now including a class-I integron carrying blaVIM-1 and other resistance genes. Notably, this isolate was able to conjugate the double carbapenemase plasmid to an E. coli recipient, conferring resistance to 1st -5th generation cephalosporins (including combinations with beta-lactamase inhibitors), penicillins, monobactams, and carbapenems. CONCLUSIONS: We reported the isolation in Chile of high-risk carbapenem-resistant hvKp carrying a highly transmissible conjugative plasmid encoding KPC-2 and VIM-1 carbapenemases, conferring resistance to most beta-lactams. Furthermore, the lack of hypermucoviscosity argues against this trait as a reliable hvKp marker. These findings highlight the rapid evolution towards multi-drug resistance of hvKp in Chile and globally, as well as the importance of conjugative plasmids and other mobile genetic elements in this convergence. In this regard, genomic approaches provide valuable support to monitor and obtain essential information on these priority pathogens and mobile elements.

Assuntos

RESUMO

The emergence of hypervirulent Klebsiella pneumoniae (hvKP) strains poses a significant threat to public health due to high mortality rates and propensity to cause severe community-acquired infections in healthy individuals. The ability to form biofilms and produce a protective capsule contributes to its enhanced virulence and is a significant challenge to effective antibiotic treatment. Polyphosphate kinase 1 (PPK1) is an enzyme responsible for inorganic polyphosphate synthesis and plays a vital role in regulating various physiological processes in bacteria. In this study, we investigated the impact of polyP metabolism on the biofilm and capsule formation and virulence traits in hvKP using Dictyostelium discoideum amoeba as a model host. We found that the PPK1 null mutant was impaired in biofilm and capsule formation and showed attenuated virulence in D. discoideum compared to the wild-type strain. We performed a proteomic analysis to gain further insights into the underlying molecular mechanism. The results revealed that the PPK1 mutant had a differential expression of proteins involved in capsule synthesis (Wzi-Ugd), biofilm formation (MrkC-D-H), synthesis of the colibactin genotoxin precursor (ClbB), as well as proteins associated with the synthesis and modification of lipid A (ArnB-LpxC-PagP). These proteomic findings corroborate the phenotypic observations and indicate that the PPK1 mutation is associated with impaired biofilm and capsule formation and attenuated virulence in hvKP. Overall, our study highlights the importance of polyP synthesis in regulating extracellular biomolecules and virulence in K. pneumoniae and provides insights into potential therapeutic targets for treating K. pneumoniae infections.

Assuntos

RESUMO

Background The convergence of hypervirulence and carbapenem resistance in the bacterial pathogen Klebsiella pneumoniae represents a critical global health concern. Hypervirulent K. pneumoniae (hvKp) strains, frequently from sequence type 23 (ST23) and having a K1 capsule, have been associated with severe community-acquired invasive infections. Although hvKp were initially restricted to Southeast Asia and primarily antibiotic-sensitive, carbapenem-resistant hvKp infections are reported worldwide. Here, within the carbapenemase production Enterobacterales surveillance system headed by the Chilean Public Health Institute, we describe the isolation in Chile of a high-risk ST23 dual-carbapenemase-producing hvKp strain, which carbapenemase genes are encoded in a single conjugative plasmid. Results Phenotypic and molecular tests of this strain revealed an extensive resistance to at least 15 antibiotic classes and the production of KPC-2 and VIM-1 carbapenemases. Unexpectedly, this isolate lacked hypermucoviscosity, challenging this commonly used hvKp identification criteria. Complete genome sequencing and analysis confirmed the K1 capsular type, the KpVP-1 virulence plasmid, and the GIE492 and ICEKp10 genomic islands carrying virulence factors strongly associated with hvKp. Although this isolate belonged to the globally disseminated hvKp clonal group CG23-I, it is unique, as it formed a clade apart from a previously reported Chilean ST23 hvKp isolate and acquired an IncN KPC-2 plasmid highly disseminated in South America (absent in other hvKp genomes), but now including a class-I integron carrying blaVIM−1 and other resistance genes. Notably, this isolate was able to conjugate the double carbapenemase plasmid to an E. coli recipient, conferring resistance to 1st-5th generation cephalosporins (including combinations with beta-lactamase inhibitors), penicillins, monobactams, and carbapenems. Conclusions We reported the isolation in Chile of high-risk carbapenem-resistant hvKp carrying a highly transmissible conjugative plasmid encoding KPC-2 and VIM-1 carbapenemases, conferring resistance to most beta-lactams. Furthermore, the lack of hypermucoviscosity argues against this trait as a reliable hvKp marker. These findings highlight the rapid evolution towards multi-drug resistance of hvKp in Chile and globally, as well as the importance of conjugative plasmids and other mobile genetic elements in this convergence. In this regard, genomic approaches provide valuable support to monitor and obtain essential information on these priority pathogens and mobile elements.

RESUMO

Multidrug- and carbapenem-resistant Klebsiella pneumoniae (CR-Kp) are critical threats to global health and key traffickers of resistance genes to other pathogens. Despite the sustained increase in CR-Kp infections in Chile, few strains have been described at the genomic level, lacking details of their resistance and virulence determinants and the mobile elements mediating their dissemination. In this work, we studied the antimicrobial susceptibility and performed a comparative genomic analysis of 10 CR-Kp isolates from the Chilean surveillance of carbapenem-resistant Enterobacteriaceae. High resistance was observed among the isolates (five ST25, three ST11, one ST45, and one ST505), which harbored 44 plasmids, most carrying genes for conjugation and resistance to several antibiotics and biocides. Ten plasmids encoding carbapenemases were characterized, including novel plasmids or variants with additional resistance genes, a novel genetic environment for blaKPC-2, and plasmids widely disseminated in South America. ST25 K2 isolates belonging to CG10224, a clone traced back to 2012 in Chile, which recently acquired blaNDM-1, blaNDM-7, or blaKPC-2 plasmids stood out as high-risk clones. Moreover, this corresponds to the first report of ST25 and ST45 Kp producing NDM-7 in South America and ST505 CR-Kp producing both NDM-7 and KPC-2 worldwide. Also, we characterized a variety of genomic islands carrying virulence and fitness factors. These results provide baseline knowledge for a detailed understanding of molecular and genetic determinants behind antibiotic resistance and virulence of CR-Kp in Chile and South America. IMPORTANCE In the ongoing antimicrobial resistance crisis, carbapenem-resistant strains of Klebsiella pneumoniae are critical threats to public health. Besides globally disseminated clones, the burden of local problem clones remains substantial. Although genomic analysis is a powerful tool for improving pathogen and antimicrobial resistance surveillance, it is still restricted in low- to middle-income countries, including Chile, causing them to be underrepresented in genomic databases and epidemiology surveys. This study provided the first 10 complete genomes of the Chilean surveillance for carbapenem-resistant K. pneumoniae in healthcare settings, unveiling their resistance and virulence determinants and the mobile genetic elements mediating their dissemination, placed in the South American and global K. pneumoniae epidemiological context. We found ST25 with K2 capsule as an emerging high-risk clone, along with other lineages producing two carbapenemases and several other resistance and virulence genes encoded in novel plasmids and genomic islands.

RESUMO

Piscirickettsia salmonis is the etiologic agent of piscirickettsiosis, a disease that causes significant losses in the salmon farming industry. In order to unveil the pathogenic mechanisms of P. salmonis, appropriate molecular and cellular studies in multiple cell lines with different origins need to be conducted. Toward that end, we established a cell viability assay that is suitable for high-throughput analysis using the alamarBlue reagent to follow the distinct stages of the bacterial infection cycle. Changes in host cell viability can be easily detected using either an absorbance- or fluorescence-based plate reader. Our method accurately tracked the infection cycle across two different Atlantic salmon-derived cell lines, with macrophage and epithelial cell properties, and zebrafish primary cell cultures. Analyses were also carried out to quantify intracellular bacterial replication in combination with fluorescence microscopy to visualize P. salmonis and cellular structures in fixed cells. In addition, dual gene expression analysis showed that the pro-inflammatory cytokines IL-6, IL-12, and TNFα were upregulated, while the cytokines IL1b and IFNγ were downregulated in the three cell culture types. The expression of the P. salmonis metal uptake and heme acquisition genes, together with the toxin and effector genes ospD3, ymt, pipB2 and pepO, were upregulated at the early and late stages of infection regardless of the cell culture type. On the other hand, Dot/Icm secretion system genes as well as stationary state and nutrient scarcity-related genes were upregulated only at the late stage of P. salmonis intracellular infection. We propose that these genes encoding putative P. salmonis virulence factors and immune-related proteins could be suitable biomarkers of P. salmonis infection. The infection protocol and cell viability assay described here provide a reliable method to compare the molecular and cellular changes induced by P. salmonis in other cell lines and has the potential to be used for high-throughput screenings of novel antimicrobials targeting this important fish intracellular pathogen.

RESUMO

Piscirickettsiasalmonis is an intracellular bacterial fish pathogen that causes piscirickettsiosis, a disease with numerous negative impacts in the Chilean salmon farming industry. Although transcriptomic studies of P. salmonis and its host have been performed, dual host-pathogen proteomic approaches during infection are still missing. Considering that gene expression does not always correspond with observed phenotype, and bacteriological culture studies inadequately reflect infection conditions, to improve the existing knowledge for the pathogenicity of P. salmonis, we present here a global proteomic profiling of Salmon salar macrophage-like cell cultures infected with P. salmonis LF-89. The proteomic analyses identified several P. salmonis proteins from two temporally different stages of macrophages infection, some of them related to key functions for bacterial survival in other intracellular pathogens. Metabolic differences were observed in early-stage infection bacteria, compared to late-stage infections. Virulence factors related to membrane, lipopolysaccharide (LPS) and surface component modifications, cell motility, toxins, and secretion systems also varied between the infection stages. Pilus proteins, beta-hemolysin, and the type VI secretion system (T6SS) were characteristic of the early-infection stage, while fimbria, upregulation of 10 toxins or effector proteins, and the Dot/Icm type IV secretion system (T4SS) were representative of the late-infection stage bacteria. Previously described virulence-related genes in P. salmonis plasmids were identified by proteomic assays during infection in SHK-1 cells, accompanied by an increase of mobile-related elements. By comparing the infected and un-infected proteome of SHK-1 cells, we observed changes in cellular and redox homeostasis; innate immune response; microtubules and actin cytoskeleton organization and dynamics; alteration in phagosome components, iron transport, and metabolism; and amino acids, nucleoside, and nucleotide metabolism, together with an overall energy and ATP production alteration. Our global proteomic profiling and the current knowledge of the P. salmonis infection process allowed us to propose a model of the macrophage-P. salmonis interaction.

RESUMO

In the teleost egg, the embryo is immersed in an extraembryonic fluid that fills the space between the embryo and the chorion and partially isolates it from the external environment, called the perivitelline fluid (PVF). The exact composition of the PVF remains unknown in vertebrate animals. The PVF allows the embryo to avoid dehydration, to maintain a safe osmotic balance and provides mechanical protection; however, its potential defensive properties against bacterial pathogens has not been reported. In this work, we determined the global proteomic profile of PVF in zebrafish eggs and embryos, and the maternal or zygotic origin of the identified proteins was studied. In silico analysis of PVF protein composition revealed an enrichment of protein classes associated with non-specific humoral innate immunity. We found lectins, protease inhibitors, transferrin, and glucosidases present from early embryogenesis until hatching. Finally, in vitro and in vivo experiments done with this fluid demonstrated that the PVF possessed a strong agglutinating capacity on bacterial cells and protected the embryos when challenged with the pathogenic bacteria Edwardsiella tarda. Our results suggest that the PVF is a primitive inherited immune extraembryonic system that protects the embryos from external biological threats prior to hatching.

Assuntos

RESUMO

Within the southern California Current ecosystem there are two well-documented breaks in marine community structure at Point Conception and Punta Eugenia. We explored the presence of similar breaks in a diverse zooplankton community through metabarcoding of mixed net tow tissue samples collected during an expedition from Monterey to Baja California in February of 2012. We recovered a high diversity of species as well as patterns of species presence that align with their previously documented ranges in this region. We found a clear break at Punta Eugenia in overall zooplankton community structure, while Point Conception was weakly linked to changes in community structure. We analyzed this dataset through two parallel bioinformatic pipelines to examine the robustness of these results. Our overall conclusions were consistent across both pipelines, however there were differences in species detection. This study illustrates the utility of metabarcoding analysis on mixed tissue samples for recovering known patterns of diversity, as well as allowing elucidation of broad patterns of community differentiation across many groups of organisms.

Assuntos

RESUMO

Four large cryptic plasmids were identified in the salmon pathogen Piscirickettsia salmonis reference strain LF-89. These plasmids appeared highly novel, with less than 7% nucleotidic identity to the nr plasmid database. Plasmid copy number analysis revealed that they are harbored in chromosome equivalent ratios. In addition to plasmid-related genes (plasmidial autonomous replication, partitioning, maintenance, and mobilization genes), mobile genetic elements such as transposases, integrases, and prophage sequences were also identified in P. salmonis plasmids. However, bacterial lysis was not observed upon the induction of prophages. A total of twelve putative virulence factors (VFs) were identified, in addition to two global transcriptional regulators, the widely conserved CsrA protein and the regulator Crp/Fnr. Eleven of the putative VFs were overexpressed during infection in two salmon-derived cellular infection models, supporting their role as VFs. The ubiquity of these plasmids was also confirmed by sequence similarity in the genomes of other P. salmonis strains. The ontology of P. salmonis plasmids suggests a role in bacterial fitness and adaptation to the environment as they encode proteins related to mobilization, nutrient transport and utilization, and bacterial virulence. Further functional characterization of P. salmonis plasmids may improve our knowledge regarding virulence and mobile elements in this intracellular pathogen.

RESUMO

Inorganic polyphosphate (polyP) and its metabolic enzymes are important in several cellular processes related with virulence and antibiotic susceptibility. Accordingly, bacterial polyP synthesis has been proposed as a good target for designing novel antivirulence molecules as alternative to conventional antibiotics. In most pathogenic bacteria, polyphosphate kinase 1 (PPK1), in charge of polyP synthesis from ATP, is widely conserved. Current colorimetric and radioactive polyP synthesis enzymatic assays are not suitable for high-throughput screening of PPK1 inhibitors. Given the ability of polyP to modify the excitation-emission spectra of DAPI (4'-6-diamidino-2-phenylindole), a fluorescence assay was previously developed by using a purified recombinant PPK1 enzyme from Escherichia coli. In this work we have developed a suitable methodology for high-throughput measurement of E. coli PPK1 activity. This platform can be used for the screening putative antimicrobial molecules for related enteropathogenic bacteria.

RESUMO

Piscirickettsia salmonis is the causative agent of Piscirickettsiosis, a systemic infection of salmonid fish species. P. salmonis infects and survives in its host cell, a process that correlates with the expression of virulence factors including components of the type IVB secretion system. To gain further insights into the cellular and molecular mechanism behind the adaptive response of P. salmonis during host infection, we established an in vitro model of infection using the SHK-1 cell line from Atlantic salmon head kidney. The results indicated that in comparison to uninfected SHK-1 cells, infection significantly decreased cell viability after 10 days along with a significant increment of P. salmonis genome equivalents. At that time, the intracellular bacteria were localized within a spacious cytoplasmic vacuole. By using a whole-genome microarray of P. salmonis LF-89, the transcriptome of this bacterium was examined during intracellular growth in the SHK-1 cell line and exponential growth in broth. Transcriptome analysis revealed a global shutdown of translation during P. salmonis intracellular growth and suggested an induction of the stringent response. Accordingly, key genes of the stringent response pathway were up-regulated during intracellular growth as well as at stationary phase bacteria, suggesting a role of the stringent response on bacterial virulence. Our results also reinforce the participation of the Dot/Icm type IVB secretion system during P. salmonis infection and reveals many unexplored genes with potential roles in the adaptation to intracellular growth. Finally, we proposed that intracellular P. salmonis alternates between a replicative phase and a stationary phase in which the stringent response is activated.

Assuntos

RESUMO

Important features of host-pathogen interactions have been discovered using nonmammalian hosts. Therefore, model organisms such as the nematode Caenorhabditis elegans, the social amoeba Dictyostelium discoideum, and zebrafish ( Danio rerio ) have been increasingly used for studying bacterial pathogenesis in vivo. These host models are amenable for live cell imaging studies, which can also benefit from online resources and databases ( Dictybase.org , ZFIN.org , Wormbase.org ), as well as from a wide repertoire of genetic and genomic tools generated over the years by the scientific community. Here, we present the protocols we developed to study bacterial dynamics within infected embryonic zebrafish. This chapter describes detailed methods to achieve infections of zebrafish larvae with the foodborne pathogen Salmonella enterica serovar Typhimurium, including embryonic zebrafish spawning and maintenance, bacterial inoculation through intravenous injections and static immersion, followed by fluorescence imaging of infected transgenic zebrafish. Methods for studying bacterial dynamics within zebrafish larvae through live cell imaging are also described.

Assuntos

RESUMO

We present a protocol for the study of inter and transgenerational behavioral responses to pathogenesis in C. elegans. Transgenerational and intergenerational effects of microbes are best studied in model organisms with short life cycles, large progenies, and quantifiable cellular and behavioral outcomes. This chapter encompasses basic techniques used to study the consequences of bacterial infection in C. elegans, including worm growth, quantification of dauer larvae, and quantification of bacterial population dynamics within individual animals. Specific methods for studying transgenerational effects and their duration are also described.

Assuntos

RESUMO

Inhibiting pathogenic bacterial adherence on surfaces is an ongoing challenge to prevent the development of biofilms. Multilayer polyelectrolyte films are feasible antibacterial materials. Here, we have designed new films made of carbohydrate polyelectrolytes to obtain antibacterial coatings that prevent biofilm formation. The polyelectrolyte films were constructed from poly(maleic anhydride- alt-styrene) functionalized with glucofuranose derivatives and quaternized poly(4-vinylpyridine) N-alkyl. These films prevent Pseudomonas aeruginosa and Salmonella Typhimurium, two important bacterial contaminants in clinical environments, from adhering to surfaces. When the film was composed of more than 10 layers, the bacterial population was greatly reduced, while the bacteria remaining on the film were morphologically damaged, as atomic force microscopy revealed. The antibacterial capacity of the polyelectrolyte films was determined by the combination of thickness, wettability, surface energy, and most importantly, the conformation that polyelectrolytes adopt the function of nature of the carbohydrate group. This polyelectrolyte film constitutes the first green approach to preventing pathogenic bacterial surface adherence and proliferation without killing the bacterial pathogen.

Assuntos

RESUMO

Inorganic polyphosphate (polyP) deficiency in enteric bacterial pathogens reduces their ability to invade and establish systemic infections in different hosts. For instance, inactivation of the polyP kinase gene (ppk) encoding the enzyme responsible for polyP biosynthesis reduces invasiveness and intracellular survival of Salmonella enterica serovar Typhimurium (S. Typhimurium) in epithelial cells and macrophages in vitro. In addition, the virulence in vivo of a S. Typhimurium Δppk mutant is significantly reduced in a murine infection model. In spite of these observations, the role played by polyP during the Salmonella-host interaction is not well understood. The social amoeba Dictyostelium discoideum has proven to be a useful model for studying relevant aspects of the host-pathogen interaction. In fact, many intracellular pathogens can survive within D. discoideum cells using molecular mechanisms also required to survive within macrophages. Recently, we established that S. Typhimurium is able to survive intracellularly in D. discoideum and identified relevant genes linked to virulence that are crucial for this process. The aim of this study was to determine the effect of a polyP deficiency in S. Typhimurium during its interaction with D. discoideum. To do this, we evaluated the intracellular survival of wild-type and Δppk strains of S. Typhimurium in D. discoideum and the ability of these strains to delay the social development of the amoeba. In contrast to the wild-type strain, the Δppk mutant was unable to survive intracellularly in D. discoideum and enabled the social development of the amoeba. Both phenotypes were complemented using a plasmid carrying a copy of the ppk gene. Next, we simultaneously evaluated the proteomic response of both S. Typhimurium and D. discoideum during host-pathogen interaction via global proteomic profiling. The analysis of our results allowed the identification of novel molecular signatures that give insight into Salmonella-Dictyostelium interaction. Altogether, our results indicate that inorganic polyP is essential for S. Typhimurium virulence and survival in D. discoideum. In addition, we have validated the use of global proteomic analyses to simultaneously evaluate the host-pathogen interaction of S. Typhimurium and D. discoideum. Furthermore, our infection assays using these organisms can be exploited to screen for novel anti-virulence molecules targeting inorganic polyP biosynthesis.

Assuntos

RESUMO

Multiresistant and invasive hypervirulent Klebsiella pneumoniae strains have become one of the most urgent bacterial pathogen threats. Recent analyses revealed a high genomic plasticity of this species, harboring a variety of mobile genetic elements associated with virulent strains, encoding proteins of unknown function whose possible role in pathogenesis have not been addressed. K. pneumoniae virulence has been studied mainly in animal models such as mice and pigs, however, practical, financial, ethical and methodological issues limit the use of mammal hosts. Consequently, the development of simple and cost-effective experimental approaches with alternative host models is needed. In this work we described the use of both, the social amoeba and professional phagocyte Dictyostelium discoideum and the fish Danio rerio (zebrafish) as surrogate host models to study K. pneumoniae virulence. We compared three K. pneumoniae clinical isolates evaluating their resistance to phagocytosis, intracellular survival, lethality, intestinal colonization, and innate immune cells recruitment. Optical transparency of both host models permitted studying the infective process in vivo, following the Klebsiella-host interactions through live-cell imaging. We demonstrated that K. pneumoniae RYC492, but not the multiresistant strains 700603 and BAA-1705, is virulent to both host models and elicits a strong immune response. Moreover, this strain showed a high resistance to phagocytosis by D. discoideum, an increased ability to form biofilms and a more prominent and irregular capsule. Besides, the strain 700603 showed the unique ability to replicate inside amoeba cells. Genomic comparison of the K. pneumoniae strains showed that the RYC492 strain has a higher overall content of virulence factors although no specific genes could be linked to its phagocytosis resistance, nor to the intracellular survival observed for the 700603 strain. Our results indicate that both zebrafish and D. discoideum are advantageous host models to study different traits of K. pneumoniae that are associated with virulence.

Assuntos

RESUMO

The dynamic response of organisms exposed to environmental pathogens determines their survival or demise, and the outcome of this interaction depends on the host's susceptibility and pathogen-dependent virulence factors. The transmission of acquired information about the nature of a pathogen to progeny may ensure effective defensive strategies for the progeny's survival in adverse environments. Environmental RNA interference (RNAi) is a systemic and heritable mechanism and has recently been linked to antibacterial and antifungal defenses in both plants and animals. Here, we report that the second generation of Caenorhabditis elegans living on pathogenic bacteria can avoid bacterial infection by entering diapause in an RNAi pathway-dependent mechanism. Furthermore, we demonstrate that the information encoding this survival strategy is transgenerationally transmitted to the progeny via the maternal germ line.IMPORTANCE Bacteria vastly influence physiology and behavior, and yet, the specific mechanisms by which they cause behavioral changes in hosts are not known. We use C. elegans as a host and the bacteria they eat to understand how microbes trigger a behavioral change that helps animals to survive. We found that animals faced with an infection for two generations could enter a hibernationlike state, arresting development by forming dauer larvae. Dauers have closed mouths and effectively avoid infection. Animals accumulate information that is transgenerationally transmitted to the next generations to form dauers. This work gives insight on how bacteria communicate in noncanonical ways with their hosts, resulting in long-lasting effects providing survival strategies to the community.

Assuntos