Protocol for live imaging of bacteria-cell interactions in genetically modified mouse small intestinal organoids.

Here, we present a protocol for microinjection of bacteria into mouse small intestinal organoids that recapitulates the natural route of infection of intestinal epithelial cells from the intestinal lumen. We describe steps for visualizing bacteria-cell interactions by live imaging of infected organoids using light sheet microscopy. We then detail procedures for generating doxycycline-inducible expression of mutant proteins in organoids to study essential gene functions. The different techniques described in this protocol can be used independently as required. For complete details on the use and execution of this protocol, please refer to Kim et al. (2021).1.

Live Imaging Reveals Listeria Hijacking of E-Cadherin Recycling as It Crosses the Intestinal Barrier.

Listeria monocytogenes is a foodborne bacterial pathogen that causes human listeriosis, a severe systemic infection.1 Its translocation across the intestinal epithelium is mediated by the interaction of internalin (InlA), a Listeria surface protein, with its host-species-specific receptor E-cadherin (Ecad).2-5 It occurs through goblet cells, on which Ecad is luminally accessible,6 via an unknown mechanism. In the absence of cell lines recapitulating this phenotype in vitro, we developed an ex vivo experimental system, based on the intraluminal microinjection of Listeria in untreated, pharmacologically treated, and genetically modified intestinal organoids. Using both live light-sheet microscopy and confocal imaging, we show that Listeria translocates through goblet cells within a membrane vacuole in an InlA- and microtubule-dependent manner. As Ecad undergoes constant apical-basal recycling,7,8 we hypothesized that Lm may transit through goblet cells by hijacking Ecad recycling pathway. Indeed, Listeria is stuck at goblet cell apex when Ecad endocytosis is blocked and remains trapped intracellularly at the basolateral pole of goblet cells when Rab11-dependent Ecad recycling is compromised. Together, these results show that Listeria, upon docking onto its luminally accessible receptor Ecad, hijacks its recycling pathway to be transferred by transcytosis across goblet cells. Live imaging of host-pathogen interactions in organoids is a promising approach to dissect their underlying cell and molecular biology.

Author Correction: Hypervirulent Listeria monocytogenes clones' adaptation to mammalian gut accounts for their association with dairy products.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Hypervirulent Listeria monocytogenes clones' adaption to mammalian gut accounts for their association with dairy products.

Listeria monocytogenes (Lm) is a major human and animal foodborne pathogen. Here we show that hypervirulent Lm clones, particularly CC1, are strongly associated with dairy products, whereas hypovirulent clones, CC9 and CC121, are associated with meat products. Clone adaptation to distinct ecological niches and/or different food products contamination routes may account for this uneven distribution. Indeed, hypervirulent clones colonize better the intestinal lumen and invade more intestinal tissues than hypovirulent ones, reflecting their adaption to host environment. Conversely, hypovirulent clones are adapted to food processing environments, with a higher prevalence of stress resistance and benzalkonium chloride tolerance genes and a higher survival and biofilm formation capacity in presence of sub-lethal benzalkonium chloride concentrations. Lm virulence heterogeneity therefore reflects the diversity of the ecological niches in which it evolves. These results also have important public health implications and may help in reducing food contamination and improving food consumption recommendations to at-risk populations.

Atypical Hemolytic Listeria innocua Isolates Are Virulent, albeit Less than Listeria monocytogenes.

Listeria innocua is considered a nonpathogenic Listeria species. Natural atypical hemolytic L. innocua isolates have been reported but have not been characterized in detail. Here, we report the genomic and functional characterization of representative isolates from the two known natural hemolytic L. innocua clades. Whole-genome sequencing confirmed the presence of Listeria pathogenicity islands (LIPI) characteristic of Listeria monocytogenes species. Functional assays showed that LIPI-1 and inlA genes are transcribed, and the corresponding gene products are expressed and functional. Using in vitro and in vivo assays, we show that atypical hemolytic L. innocua is virulent, can actively cross the intestinal epithelium, and spreads systemically to the liver and spleen, albeit to a lesser degree than the reference L. monocytogenes EGDe strain. Although human exposure to hemolytic L. innocua is likely rare, these findings are important for food safety and public health. The presence of virulence traits in some L. innocua clades supports the existence of a common virulent ancestor of L. monocytogenes and L. innocua.

Molecular Individual-Based Approach on Triatoma brasiliensis: Inferences on Triatomine Foci, Trypanosoma cruzi Natural Infection Prevalence, Parasite Diversity and Feeding Sources.

We used an individual-based molecular multisource approach to assess the epidemiological importance of Triatoma brasiliensis collected in distinct sites and ecotopes in Rio Grande do Norte State, Brazil. In the semi-arid zones of Brazil, this blood sucking bug is the most important vector of Trypanosoma cruzi--the parasite that causes Chagas disease. First, cytochrome b (cytb) and microsatellite markers were used for inferences on the genetic structure of five populations (108 bugs). Second, we determined the natural T. cruzi infection prevalence and parasite diversity in 126 bugs by amplifying a mini-exon gene from triatomine gut contents. Third, we identified the natural feeding sources of 60 T. brasiliensis by using the blood meal content via vertebrate cytb analysis. Demographic inferences based on cytb variation indicated expansion events in some sylvatic and domiciliary populations. Microsatellite results indicated gene flow between sylvatic and anthropic (domiciliary and peridomiciliary) populations, which threatens vector control efforts because sylvatic population are uncontrollable. A high natural T. cruzi infection prevalence (52-71%) and two parasite lineages were found for the sylvatic foci, in which 68% of bugs had fed on Kerodon rupestris (Rodentia: Caviidae), highlighting it as a potential reservoir. For peridomiciliary bugs, Galea spixii (Rodentia: Caviidae) was the main mammal feeding source, which may reinforce previous concerns about the potential of this animal to link the sylvatic and domiciliary T. cruzi cycles.

Clonogrouping, a Rapid Multiplex PCR Method for Identification of Major Clones of Listeria monocytogenes.

Three multiplex PCR assays were developed to identify the 11 most common Listeria monocytogenes clones in clinical and food samples; 270 (95.7%) of 282 strains of serogroups IVb, IIb, IIa, and IIc were identified accurately. This novel tool is a rapid and efficient alternative to multilocus sequence typing for identification of L. monocytogenes clones.

Molecular Markers and In Vitro Susceptibility to Doxycycline in Plasmodium falciparum Isolates from Thailand.

Determinations of doxycycline 50% inhibitory concentrations (IC50) for 620 isolates from northwest Thailand were performed via the isotopic method, and the data were analyzed by the Bayesian method and distributed into two populations (mean IC50s of 13.15 µM and 31.60 µM). There was no significant difference between the group with low IC50s versus the group with high IC50s with regard to copy numbers of the Plasmodium falciparum tetQ (pftetQ) gene (P = 0.11) or pfmdt gene (P = 0.87) or the number of PfTetQ KYNNNN repeats (P = 0.72).

PftetQ and pfmdt copy numbers as predictive molecular markers of decreased ex vivo doxycycline susceptibility in imported Plasmodium falciparum malaria.

BACKGROUND: The objective of this study was to evaluate the distribution of a series of independent doxycycline inhibitory concentration 50% (IC50) values to validate the trimodal distribution previously described and to validate the use of the pftetQ and pfmdt genes as molecular markers of decreased in vitro doxycycline susceptibility in Plasmodium falciparum malaria. METHODS: Doxycycline IC50 values, from 484 isolates obtained at the French National Reference Centre for Imported Malaria (Paris) between January 2006 and December 2010, were analysed for the first time by a Bayesian mixture modelling approach to distinguish the different in vitro phenotypic groups by their IC50 values. Quantitative real-time polymerase chain reaction was used to evaluate the pftetQ and pfmdt copy numbers of 89 African P. falciparum isolates that were randomly chosen from the phenotypic groups. RESULTS: The existence of at least three doxycycline phenotypes was demonstrated. The mean doxycycline IC50 was significantly higher in the group with a pftetQ copy number >1 compared to the group with a pftetQ copy number = 1 (33.17 µM versus 17.23 µM) and the group with a pfmdt copy number >1 (28.28 µM versus 16.11 µM). There was a significant difference between the combined low and medium doxycycline IC50 group and the high IC50 group in terms of the per cent of isolates with one or more copy numbers of the pftetQ gene (0% versus 20.69%) or pfmdt gene (8.33% versus 37.93%). In the logistic regression model, the pfmdt and pftetQ copy numbers >1 (odds ratio = 4.65 and 11.47) were independently associated with the high IC50 group. CONCLUSIONS: Copy numbers of pftetQ and pfmdt are potential predictive molecular markers of decreased susceptibility to doxycycline.