Deletion of the membrane protein Lmo0412 increases the virulence of Listeria monocytogenes

Methods in Molecular Biology

Sachse

Balestrino

et al. 2016

Listeria monocytogenes is a bacterial pathogen which invades and multiplies within non-professional phagocytes. Signaling cascades involved in cellular entry have been extensively analyzed, but the events leading to vacuolar escape remain less clear. In this chapter, we detail a microscopy FRET-based assay which allows quantitatively measuring L. monocytogenes infection and escape from its internalization vacuole, as well as a correlative light/electron microscopy method to investigate the morphological features of the vacuolar compartments containing L. monocytogenes.

Section: Methodsmentioning

confidence: 99%

“…We recommend to use a hard formula, which facilitates easier detachment of the coverslip. 24. It is important to have the area to be sectioned as small as possible.…”

mentioning

confidence: 99%

Assessing Vacuolar Escape of Listeria Monocytogenes

Methods in Molecular Biology

Sachse

Balestrino

et al. 2016

“…The interaction between Rli27 and the long 5′-UTR of lmo0514 is predicted to open the Shine-Dalgarno site to facilitate Lmo0514 protein translation (Quereda et al, 2014). Rli27 has also been claimed to regulate in cis lmo0412 , a gene encoding a membrane protein required for virulence (Quereda and Pucciarelli, 2014). Another recent study reported that the multicopy sRNA LhrC is induced by L. monocytogenes in the intracellular environment and in response to envelope stress (Sievers et al, 2014).…”

Section: Examples Of Regulation By Non-coding Rnas In Intracellular Bmentioning

confidence: 99%

“…As a representative example, ~ 3 × 10 7 fibroblasts and a MOI of 50:1 (bacteria:cell) were infected with S. Typhimurium to obtain sufficient RNA for qRT-PCR assays (Ortega et al, 2012). In another study, ~ 6 × 10 7 epithelial cells were infected at a MOI of 10:1 (bacteria: cell) with L. monocytogenes to obtain RNA for northern blotting assays (Quereda and Pucciarelli, 2014). …”

Section: Studying Gene Expression In Intracellular Bacteriamentioning

confidence: 99%

Non-coding RNA regulation in pathogenic bacteria located inside eukaryotic cells

Ortega

Front. Cell. Infect. Microbiol.

Pucciarelli

et al. 2014

Intracellular bacterial pathogens have evolved distinct lifestyles inside eukaryotic cells. Some pathogens coexist with the infected cell in an obligate intracellular state, whereas others transit between the extracellular and intracellular environment. Adaptation to these intracellular lifestyles is regulated in both space and time. Non-coding small RNAs (sRNAs) are post-transcriptional regulatory molecules that fine-tune important processes in bacterial physiology including cell envelope architecture, intermediate metabolism, bacterial communication, biofilm formation, and virulence. Recent studies have shown production of defined sRNA species by intracellular bacteria located inside eukaryotic cells. The molecules targeted by these sRNAs and their expression dynamics along the intracellular infection cycle remain, however, poorly characterized. Technical difficulties linked to the isolation of “intact” intracellular bacteria from infected host cells might explain why sRNA regulation in these specialized pathogens is still a largely unexplored field. Transition from the extracellular to the intracellular lifestyle provides an ideal scenario in which regulatory sRNAs are intended to participate; so much work must be done in this direction. This review focuses on sRNAs expressed by intracellular bacterial pathogens during the infection of eukaryotic cells, strategies used with these pathogens to identify sRNAs required for virulence, and the experimental technical challenges associated to this type of studies. We also discuss varied techniques for their potential application to study RNA regulation in intracellular bacterial infections.

“…To date, no study has focused on changes at the proteome level when L. monocytogenes copes with the host defenses present in the blood. This is relevant considering that L. monocytogenes remodels at a large extent the cell wall proteome when it resides inside the eukaryotic cell (Garcia‐del Portillo et al ., ; Quereda et al ., ; Quereda and Pucciarelli, ). Following the transit from the extracellular environment to the eukaryotic intracellular niche, L. monocytogenes up‐regulates surface proteins such as the membrane protein ActA, which promotes actin recruitment, and the peptidoglycan‐bound proteins Internalin A (Inl‐A) and Lmo0514 (Quereda et al ., ).…”

Section: Introductionmentioning

confidence: 98%

Listeria monocytogenes remodels the cell surface in the blood‐stage

Portillo

Pucciarelli

2016

Environ Microbiol Rep

After crossing the intestinal barrier, the bacterial pathogen Listeria monocytogenes disseminates via the blood to the liver, spleen, brain and placenta. Transcriptomic studies have shown that L. monocytogenes changes expression of many genes during this blood-stage. However, no comparable data at the protein level are known. As main interactors with the environment, we focused in surface proteins produced by L. monocytogenes in an ex vivo bovine blood model. Bacteria exposed to blood alter selectively the amount of several surface proteins compared with bacteria grown in laboratory media. Increased levels were detected for Lmo0514 and Internalin A, two surface proteins covalently bound to peptidoglycan, and the moonlighting protein alcohol-acetaldehyde dehydrogenase, also known as Lap for 'Listeria adhesion protein'. Lmo0514, induced by L. monocytogenes inside epithelial cells, is required for survival in plasma and for virulence in mice at early infection stages. Lmo0514 is also important to cope with low pH stress. By contrast, L. monocytogenes down-regulates other surface proteins following exposure to blood and plasma such as Internalin I. These data provide evidence for remodelling of the L. monocytogenes cell surface during the blood-stage, which it could facilitate pathogen dissemination to deep organs.