Xenophagy, also known as antibacterial autophagy, plays a role in host defence against invading pathogens such as Group A Streptococcus (GAS) and Salmonella. In xenophagy, autophagy receptors are used in the recognition of invading pathogens and in autophagosome maturation and autolysosome formation. However, the mechanism by which autophagy receptors are regulated during bacterial infection remains poorly elucidated. In this study, we identified LAMTOR2 and LAMTOR1, also named p14 and p18, respectively, as previously unrecognised xenophagy regulators that modulate the autophagy receptor TAX1BP1 in response to GAS and Salmonella invasion. LAMTOR1 was localized to bacterium‐containing endosomes, and LAMTOR2 was recruited to bacterium‐containing damaged endosomes in a LAMTOR1‐dependent manner. LAMTOR2 was dispensable for the formation of autophagosomes targeting damaged membrane debris surrounding cytosolic bacteria, but it was critical for autolysosome formation, and LAMTOR2 interacted with the autophagy receptors NBR1, TAX1BP1, and p62 and was necessary for TAX1BP1 recruitment to pathogen‐containing autophagosomes. Notably, knockout of TAX1BP1 caused a reduction in autolysosome formation and subsequent bacterial degradation. Collectively, our findings demonstrated that the LAMTOR1/2 complex is required for recruiting TAX1BP1 to autophagosomes and thereby facilitating autolysosome formation during bacterial infection.
The permeability barrier of skin resides in the stratum corneum, and its properties are mediated by a series of lipid multilayers, enriched in ceramides, cholesterol, and free fatty acids, segregated within the stratum corneum (SC) interstices. SC lipid content is usually determined by gravimetric methods in conjunction with high performance thin layer chromatography, but these methods are time‒consuming and involve hazardous solvents. The objective of the present study was to develop a method of measuring SC lipid content by Fourier transform infrared spectrometry (FTIR) that is fast and requires no solvents. The IR spectra of isolated porcine SC sheets were recorded using a FTIR spectrometer. SC lipid content was determined by gravimetric methods using chloroform–methanol extraction. The peak area of both the CH2symmetric (2850 cm−1) and asymmetric (2920 cm−1) stretching bands in the IR spectra of progressively solvent‒extracted porcine SC sheets decreased with increasing amount of SC lipids removed. When spectral analysis was performed by curve‒fitting using GRAMS/32 software between 3000 to 2800 cm−1, peak area ratios of CH2to CH3asymmetric stretching bands in the IR spectra of 46 isolated porcine SC samples were correlated to SC lipid content (R2=0.90), with the standard error of measurement of 1.91%. The study demonstrated the feasibility of using FTIR technique to rapidly and accurately measure SC lipid content.
Group A Streptococcus (GAS) invades epithelial cells causing persistent infection. GAS has a variety of effector proteins that modulate host systems to affect their survival in host environments. The main effector proteins of GAS are NAD-glycohydrolase (Nga) and streptolysin O (SLO). Although Nga has NADase activity and shows SLO-dependent cytotoxicity, some clinical isolates harbor NADase-inactive subtypes of Nga, and the function of NADase-inactive Nga is still unclear. In this study, we found that deletion of nga enhanced the internalization of GAS into HeLa and Ca9-22 cells. Amino acid substitution of Nga R289K/G330D (NADase-inactive) does not enhance GAS invasion, suggesting that Nga may inhibit the internalization of GAS into host cells in an NADase-independent manner. Moreover, double deletion of slo and nga showed similar invasion percentages compared with wild-type GAS, indicating the important role of SLO in the inhibition of GAS invasion by Nga. Furthermore, enhanced internalization of the nga deletion mutant was not observed in Cav1-knockout HeLa cells. Altogether, these findings demonstrate an unrecognized NADase-independent function of Nga as a negative regulator of CAV1-mediated internalization into epithelial cells.
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