Autophagy-independent function of MAP-LC3 during intracellular propagation of Chlamydia trachomatis.

Microtubule-associated protein 1 (MAP1) light chain 3 (LC3) has proven useful as autophagosomal marker in studies on the interaction between pathogens and the host autophagic machinery. However, the function of LC3 is known to extend above and beyond its role in autophagosome formation. We previously reported that intrinsic LC3 is associated with the intracellular Chlamydia trachomatis inclusion in human epithelial cells. Here we show that LC3, most likely the cytoplasmic nonlipidated form, interacts with the C. trachomatis inclusion as a microtubule-associated protein rather than an autophagosome-associated component. In contrast, N-terminally GFP-tagged LC3 exclusively targets autophagosomes rather than chlamydial inclusions. Immunofluorescence analysis revealed an association of LC3 and MAP1 subunits A and B with the inclusion as early as 18 h post infection. Inclusion-bound LC3 was connected with the microtubular network. Depolymerization of the microtubular architecture disrupted the association of LC3/MAP1s with the inclusion. Furthermore, siRNA-mediated silencing of the MAP1 and LC3 proteins revealed their essential function in the intracellular growth of C. trachomatis. Interestingly, defective autophagy remarkably enhanced chlamydial growth, suggesting a suppressive effect of the autophagic machinery on bacterial development. However, depletion of LC3 in autophagy-deficient cells noticeably reduced chlamydial propagation. Thus, our findings demonstrate a new function for LC3, distinct from autophagy, in intracellular bacterial pathogenesis.

Long-term effects of natural amino acids on infection with Chlamydia trachomatis.

Supplementation of culture media with leucine, isoleucine, methionine, or phenylalanine was previously found to inhibit Chlamydia trachomatis growth in HEp-2 cells. Here, we investigated the long-term effects of these additives on C. trachomatis infection in the same cell model. Amino acid addition 30h post-infection (pi) effectively suppressed the generation of infectious progeny monitored for 10 days pi. With the exception of phenylalanine, amino acid treatment beginning at 2h pi for up to 15 days led to a complete lack of infectious progeny. Phenylalanine treatment resulted in residual minimal infectivity. In extended supplementation experiments, very small aberrant chlamydial inclusions formed, whose numbers decreased considerably over time, and the production of infectious chlamydiae could not be rescued even upon amino acid withdrawal. Interestingly, a state of chlamydial persistence was induced under these conditions, as 16S rRNA transcripts were detected throughout treatment. However, expression of several key chlamydial genes including omp1, groEL, omcB, and those functioning for chlamydial DNA replication and cytokinesis was generally very low or even undetected, particularly in monolayers treated with Leu, Ile, or Met. These data revealed a capacity of certain amino acids to eliminate infectious chlamydial progeny. Additionally, supplementation of certain amino acids resulted in the formation of a small persistent population. Extrapolating from these findings may help formulate an anti-chlamydial treatment based on nutritional elements.

Competitive inhibition of amino acid uptake suppresses chlamydial growth: involvement of the chlamydial amino acid transporter BrnQ.

Chlamydiaceae are obligate intracellular bacterial pathogens that strictly depend on host metabolites, such as nucleotides, lipids, and amino acids. Depletion of amino acids in cell culture media results in abnormal chlamydial development in vitro. Surprisingly, enrichment of certain amino acids also retards chlamydial growth. Our experiments revealed that the antichlamydial effects are largely independent of changes in the host cell transcriptome or proteome and in the major signal transduction pathway modulated by amino acids, the mTOR (mammalian target of rapamycin) pathway. Furthermore, the chlamydial growth inhibition induced by leucine, isoleucine, methionine, or phenylalanine was completely reversed by concomitant addition of valine. In contrast, the growth inhibition induced by serine, glycine, or threonine was not reversed by valine addition. Functional characterization of the only predicted chlamydial transporter for branched-chain amino acids, BrnQ, revealed that it can be blocked by leucine, isoleucine, methionine, or phenylalanine but not by serine, glycine, or threonine. This chlamydial transporter is the only known BrnQ homolog possessing specificity for methionine, suggesting a unique strategy for methionine uptake among gram-negative bacteria. The antichlamydial effects of leucine, isoleucine, methionine, and phenylalanine could be explained as competitive inhibition of the BrnQ transporter and subsequent valine starvation.

Naturally occurring amino acids differentially influence the development of Chlamydia trachomatis and Chlamydia (Chlamydophila) pneumoniae.

The differential influence of individual amino acids on the growth of Chlamydia trachomatis versus Chlamydia (Chlamydophila) pneumoniae was investigated. Certain essential amino acids added in excess at the middle of the infection course resulted in varying degrees of abnormality in the development of the two species. If amino acids were added as early as 2 h post-infection, these effects were even more pronounced. The most effective amino acids in terms of C. trachomatis growth inhibition were leucine, isoleucine, methionine and phenylalanine. These amino acids elicited similar effects against C. pneumoniae, except methionine, which, surprisingly, showed a lower inhibitory activity. Tryptophan and valine marginally inhibited C. trachomatis growth and, paradoxically, led to a considerable enhancement of C. pneumoniae growth. On the other hand, some non-essential amino acids administered at the middle of or throughout the infection course differentially affected the development of the two species. For example, C. trachomatis growth was efficiently inhibited by glycine and serine, whereas C. pneumoniae was relatively less sensitive to these agents. Another difference was apparent for glutamate, glutamine and aspartate, which stimulated C. pneumoniae growth more than that of C. trachomatis. Overall, several distinctive patterns of susceptibility to excess amino acid levels were revealed for two representative C. trachomatis and C. pneumoniae isolates. Perturbation of amino acid levels, e.g. of leucine and isoleucine, might form a basis for the development of novel treatment or preventive regimens for chlamydial diseases.