A Proteomic Signature of Dormancy in the Actinobacterium Micrococcus luteus

Abstract

Dormancy is a protective state in which diverse bacteria, including Mycobacterium tuberculosis, Staphylococcus aureus, Treponema pallidum (syphilis), and Borrelia burgdorferi (Lyme disease), curtail metabolic activity to survive external stresses, including antibiotics. Evidence suggests dormancy consists of a continuum of interrelated states, including viable but nonculturable (VBNC) and persistence states. VBNC and persistence contribute to antibiotic tolerance, reemergence from latent infections, and even quorum sensing and biofilm formation. Previous studies indicate that the protein mechanisms regulating persistence and VBNC states are not well understood. We have queried the VBNC state of Micrococcus luteus NCTC 2665 (MI-2665) by quantitative proteomics combining gel electrophoresis, high-performance liquid chromatography, and tandem mass spectrometry to elucidate some of these mechanisms. MI-2665 is a nonpathogenic actinobacterium containing a small (2.5-Mb), high-GC-content genome which exhibits a well-defined VBNC state induced by nutrient deprivation. The MI-2665 VBNC state demonstrated a loss of protein diversity accompanied by increased levels of 18 proteins that are conserved across actinobacteria, 14 of which have not been previously identified in VNBC. These proteins implicate an anaplerotic strategy in the transition to VBNC, including changes in the glyoxylate shunt, redox and amino acid metabolism, and ribosomal regulatory processes. Our data suggest that MI-2665 is a viable model for dissecting the protein mechanisms underlying the VBNC stress response and provide the first protein-level signature of this state. We expect that this protein signature will enable future studies deciphering the protein mechanisms of dormancy and identify novel therapeutic strategies effective against antibiotictolerant bacterial infections.IMPORTANCE Dormancy is a protective state enabling bacteria to survive antibiotics, starvation, and the immune system. Dormancy is comprised of different states, including persistent and viable but nonculturable (VBNC) states that contribute to the spread of bacterial infections. Therefore, it is imperative to identify how bacteria utilize these different dormancy states to survive antibiotic treatment. The objective of our research is to eliminate dormancy as a route to antibiotic tolerance by understanding the proteins that control dormancy in Micrococcus luteus NCTC 2665. This bacterium has unique advantages for studying dormancy, including a small genome and a well-defined and reproducible VBNC state. Our experiments implicate four previously identified and 14 novel proteins upregulated in VBNC that may regulate this critical survival mechanism.KEYWORDS antibiotic tolerance, proteomics, stress response, systems biology D ormancy is a remarkable stress response in which bacteria curtail metabolic activity to survive potentially fatal external stresses. Dormancy has broad implications for many pathogenic bacteria, including Mycobacterium tuberculosis, Staphylococcus ...

Relationship between the Viable but Nonculturable State and Antibiotic Persister Cells

“…The VBNC state describes a subpopulation of bacteria that have transiently lost their ability to grow on routine laboratory media on which they were previously able to grow. These cells exhibit significantly lower, although present, metabolic activity than their actively growing counterparts, and they continue to maintain membrane integrity and produce proteins (10,11). In Fig.…”

“…As suggested above, it is important to recognize that neither VBNC cells nor persisters are metabolically inept. Although there is a large decline in metabolic activity in these cells, they continue to energize their membranes and produce a more specialized set of proteins (10,79). There are several mechanisms by which persister cells are thought to form, and there is growing evidence that these same mechanisms are involved in directing the VBNC state.…”

Contribution of Fluorescence Techniques in Determining the Efficiency of the Non-thermal Plasma Treatment

“…However, they can grow and divide again when culture conditions are more favorable for their metabolic recovery as a consequence the bacteria can thereafter express their pathogenicity again ( Emerson et al, 2017 ). Thus VBNC bacteria represent a significant risk in many domains such as medical field ( Passerat et al, 2009 ; Dinu and Bach, 2011 ; Zandri et al, 2012 ; Mali et al, 2017 ; Lee and Bae, 2017 ).…”

Antibiotic tolerance is associated with a broad and complex transcriptional response inE. coli

“…As expected, there was an increase in the expression of many genes regulated by sigma factors related to stationary phase and heat shock, which have previously been associated with antibiotic tolerance and cellular stress response pathways [25][26] . Namely, RpoH (σ 32 ; associated with heat shock 41 ), RpoS (σ 38 ; associated with stationary phase and stress 42 ), and RpoE (σ 24 ; also associated with heat shock 43 ) had more active regulons in the treated compared to the untreated population. Our analysis shows that RpoH transcribes many genes related to one of the COG groups noted earlier, 'posttranslational modification, protein turnover and chaperones'.…”

“…Many studies suggest that persisters are metabolically repressed, particularly with respect to translation, while others propose that active metabolism plays a vital role in persistence [16][17][18][19][20] . A few studies have leveraged RNA-sequencing (RNA-seq) [20][21][22] and proteomic data [23][24][25] , and they have identified several individual genes and systems related to antibiotic tolerance. Sigma factors and transcription factors, regulatory proteins that affect gene expression, are two classes of protein in which many individual genes have been related to antibiotic stress 15,[25][26][27][28] .…”