The bioluminescent Listeria monocytogenes strain Xen32 is defective in flagella expression and highly attenuated in orally infected BALB/cJ mice
© Bergmann et al.; licensee BioMed Central Ltd. 2013
Received: 1 May 2013
Accepted: 28 June 2013
Published: 15 July 2013
In vivo bioluminescence imaging (BLI) is a powerful method for the analysis of host-pathogen interactions in small animal models. The commercially available bioluminescent Listeria monocytogenes strain Xen32 is commonly used to analyse immune functions in knockout mice and pathomechanisms of listeriosis.
To analyse and image listerial dissemination after oral infection we have generated a murinised Xen32 strain (Xen32-mur) which expresses a previously described mouse-adapted internalin A. This strain was used alongside the Xen32 wild type strain and the bioluminescent L. monocytogenes strains EGDe-lux and murinised EGDe-mur-lux to characterise bacterial dissemination in orally inoculated BALB/cJ mice. After four days of infection, Xen32 and Xen32-mur infected mice displayed consistently higher rates of bioluminescence compared to EGDe-lux and EGDe-mur-lux infected animals. However, surprisingly both Xen32 strains showed attenuated virulence in orally infected BALB/c mice that correlated with lower bacterial burden in internal organs at day 5 post infection, smaller losses in body weights and increased survival compared to EGDe-lux or EGDe-mur-lux inoculated animals. The Xen32 strain was made bioluminescent by integration of a lux-kan transposon cassette into the listerial flaA locus. We show here that this integration results in Xen32 in a flaA frameshift mutation which makes this strain flagella deficient.
The bioluminescent L. monocytogenes strain Xen32 is deficient in flagella expression and highly attenuated in orally infected BALB/c mice. As this listerial strain has been used in many BLI studies of murine listeriosis, it is important that the scientific community is aware of its reduced virulence in vivo.
Bioluminescent in vivo imaging (BLI) of Listeria monocytogenes infections in mice has generated several new insights into the pathogenesis of listeriosis. For example, the now commercially available Listeria monocytogenes strain Xen32 was first used to demonstrate that the gallbladder is an important organ reservoir of listerial replication and pathogen shedding[1–3]. Since then the Xen32 listerial strain has been used in multiple studies as a tool to study Listeria directed immune mechanisms in knockout mice and kinetics of L. monocytogenes dissemination to target organs of listeriosis such as the bone marrow. More recently, the bioluminescent Xen32 strain has also been used to study transplacental transmission of L. monocytogenes in fetal listeriosis[6, 7].
The aim of this study was to use the bioluminescent L. monocytogenes strain Xen32 in an oral mouse listeriosis model to analyse the dissemination of the pathogen from the intestine to internal organs. To enable efficient transmission of this listerial strain through the murine gut mucosa we used our previous approach of murinisation to optimise the binding of the listerial surface protein internalin A (InlA) to the murine E-cadherin host receptor.
Material and methods
For oral inoculation of female, 9-10 weeks old BALB/cJ mice (Harlan Winkelmann, Borchen, Germany) we used our previously published mouse infection model. All experiments were conducted according to German animal welfare regulations after approval from the Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit (LAVES) as the local authority. The Listeria monocytogenes strains EGDe-lux (Lmo-EGDe-lux) and L. monocytogenes EGDe-InlA-mur-lux (Lmo-EGDe-mur-lux) have been described previously. L. monocytogenes Xen32 was purchased from Perkin Elmer (Rodgau, Germany) and genetically modified for expression of a mouse-adapted InlA as previously described. Analysis of bacterial organ counts (colony forming units, CFU) was performed as described in Bergmann et al.. BLI images were obtained using an IVIS 200 imaging system (CaliperLS) with integration time of 3 min (Xen32 strains) or 4 min (EGDe strains) at a binning of 8 and F/stop of 1. Photon flux was quantified by using the Living Image 3.1 software (CaliperLS). To assess general growth characteristics of the different L. monocytogenes strains, growth curves were performed as previously described. Luminescence was measured by quantifying photon flux of 0.5 ml culture samples from a 50 ml logarithmic L. monocytogenes culture at indicated timepoints on the IVIS 200 imaging system (5 sec integration time, binning of 8 and F/stop of 1). Genomic flaA fragments from L. monocytogenes Xen32 were amplified with flaA forward primer (5′-AGAGAAGTCTTTTCTAAACCGAATGTAGGA-3′) and flaA reverse primer (5′-CTAAGGGTAAACAATGTTCGATAAATG-3′), sequenced and analysed with MacVector 11.0.2 (MacVector Inc., Cambridge, UK). For analysis of flagella expression, listerial strains were grown overnight in BHI medium at 24°C and negatively stained with 2% uranyl acetate and examined in a Zeiss TEM910 at 80 kV. Cell invasion assays with the human colorectal epithelial cell line Caco-is deficient in flagella expression 2 (ATCC HTB-37) and the murine colon carcinoma cell line CT26 (ATCC CRL-2639) were performed as previously described. Statistical analysis of CFU data was performed using the Mann–Whitney U non-parametic test and the GraphPad Prism 5 (version 5.01) analysis software (GraphPad Software Inc.). Survival curves were statistically evaluated by Kaplan-Meier and Log- rank (Mantel-Cox) analyses.
Results and discussion
The possible roles of flagella in listeriosis pathogenesis are diverse. They include host cell adhesion and invasion, injection of virulence factors and initiation and modulation of host inflammation through recognition of flagellin by TLR5 and inflammasome receptors[16–19]. Previous studies have reported different effects of flagella deficiency on the pathogenesis of oral L. monocytogenes infections in vivo. The virulence of flagellin deletion mutants or mutants with deficiency in flagella regulatory proteins was found to be either increased or decreased depending on the listerial strain that was used for genetic modification or the mouse infection model that was employed[15, 17, 18, 20, 21]. However, these studies have used L. monocytogenes strains for oral infection challenge that were not optimized for InlA-mediated recognition of the murine E-cadherin receptor. We report here that Lmo-EGDe-lux shows increased in vivo virulence after oral infection challenge when compared to flagella deficient Lmo-Xen32 and that this difference in virulence between EGDe and Xen32 strains becomes even bigger when both strains are murinised for InlA.
The bioluminescent L. monocytogenes strain Xen32 is deficient in flagella expression and highly attenuated in virulence in an oral mouse infection model. Despite this attenuation in virulence, the L. monocytogenes strain Xen32 might be still a useful tool for in vivo imaging in experiments where sublethal or very mild infections are required (e.g. for example for phenotyping of highly susceptible or immunocompromised mouse strains). However, the scientific community should be aware that infections with L. monocytogenes Xen32 of wild type, immunocompetent mouse strains might result in smaller effects on host responses due to its in vivo virulence attenuation.
This study was supported by grants from the National German Genome Network (NGFN-Plus, grant number 01GS0855) by the European Commission under the EUMODIC project (Framework Programme 6: LSHG-CT-2006-037188) and the European COST action ‘SYSGENET’ (BM901), and Institute Strategic Grant funding from the BBSRC and the Helmholtz Centre for Infection Research (HZI). We thank Cormac Gahan (University College Cork, Ireland) for providing us the Lmo-EGDe-lux and Lmo-EGDe-mur-lux strains.
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