Group A streptococcus (GAS; Streptococcus pyogenes) is a common pathogen that invades non-phagocytic human cells via endocytosis. Once taken up by cells, it escapes from the endocytic pathway to the cytoplasm, but here it is contained within a membrane-bound structure termed GAS-containing autophagosome-like vacuoles (GcAVs). The autophagosome marker GFP-LC3 associates with GcAVs, and other components of the autophagosomal pathway are involved in GcAV formation. However, the mechanistic relationship between GcAV and canonical autophagy is largely unknown. Here, we morphologically analyzed GcAV formation in detail. Initially, a small, GFP-LC3-positive GcAV sequesters each streptococcal chain, and these then coalesce into a single, large GcAV. Expression of a dominant-negative form of Rab7 or RNAi-mediated knockdown of Rab7 prevented the initial formation of small GcAV structures. Our results demonstrate that mechanisms of GcAV formation includes not only the common machinery of autophagy, but also Rab7 as an additional component, which is dispensable in canonical autophagosome formation.

Author Summary

Autophagy has become one of the leading edge subjects in science. Autophagy occurs when a cell eats some of its cellular components and digests them. These cellular components may include cytosol and organelles as well as bacteria that has invaded the cell. Thus, autophagy plays an important role in killing pathogens. Here, we introduce an anti-bacterial autophagy called xenophagy. Group A Streptococcus (GAS) enters HeLa cells and escapes from the endosome into the cytoplasm for its growth. However, autophagy kicks in and traps GAS, thus preventing its survival path. Detailed morphological observation of this process reveals several specific features which were not found in canonical autophagy. These results provide key information about not only anti-bacterial autophagy, but also canonical autophagy.

Recombinant BchU from C. tepidum has been crystallized. Crystals diffract to 2.27 Å using synchrotron radiation at SPring-8 and belong to space group P6122 or P6522, with unit-cell parameters a = b = 81.5, c = 250.7 Å.

The S-adenosylmethionine-dependent methyltransferase BchU is an enzyme involved in the bacteriochlorophyll c biosynthetic pathway and catalyzes methylation at the C-20 position of the chlorin moiety. Recombinant Chlorobium tepidum BchU overproduced in Escherichia coli was purified and crystallized by the hanging-drop vapour-diffusion method using ammonium sulfate as a precipitant. The crystals belonged to the hexagonal space group P6122 or P6522, with unit-cell parameters a = b = 81.5, c = 250.7 Å. A native data set was collected to 2.27 Å resolution using synchrotron radiation at SPring-8.

An R-plasmid-mediated metallo-β-lactamase was found in Klebsiella pneumoniae DK4 isolated in Japan in 1991. The nucleotide sequence of its structural gene revealed that the β-lactamase termed DK4 was identical to the IMP-1 metallo-β-lactamase which was mediated by a chromosomal gene of Serratia marcescens TN9106 isolated in Japan in 1991 (E. Osano et al., Antimicrob. Agents Chemother. 38:71–78, 1994). The dose effect of DK4 β-lactamase production on the resistance levels indicated a significant contribution of the enzyme to bacterial resistance to all the β-lactams except monobactams. The enzymatic characteristics of the DK4 β-lactamase and its kinetic parameters for nine β-lactams were examined. The DK4 β-lactamase was confirmed to contain 2 mol of zinc per mol of enzyme protein. The apoenzyme that lacked the two zincs was structurally unstable, and the activities of only 30% of the apoenzyme molecules could be restored by the addition of 1 mM zinc sulfate. The substitution of five conserved histidines (His28, His86, His88, His149, His210) and a cysteine (Cys168) for an alanine indicated that His86, His88, and His149 served as ligands to one of the zincs and that Cys168 played a role as a ligand to the second zinc. Both zinc molecules contribute to the enzymatic process. Mutant enzymes that lack only one of these retained some activity. Additionally, a conserved aspartic acid at position 90 was replaced by asparagine. This mutant enzyme showed an approximately 1,000 times lower kcat value for cephalothin than that of the wild-type enzyme but retained the two zincs even after dialysis against zinc-free buffer. The observed effect of pH on the activity suggested that Asp90 functions as a general base in the enzymatic process.