Identification of secreted bacterial proteins by noncanonical amino acid tagging

Microbial quiescence and slow growth are ubiquitous physiological states, but their study is complicated by low levels of metabolic activity. To address this issue, we used a time-selective proteomelabeling method [bioorthogonal noncanonical amino acid tagging (BONCAT)] to identify proteins synthesized preferentially, but at extremely low rates, under anaerobic survival conditions by the opportunistic pathogen Pseudomonas aeruginosa. One of these proteins is a transcriptional regulator that has no homology to any characterized protein domains and is posttranscriptionally upregulated during survival and slow growth. This small, acidic protein associates with RNA polymerase, and chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing suggests that the protein associates with genomic DNA through this interaction. ChIP signal is found both in promoter regions and throughout the coding sequences of many genes and is particularly enriched at ribosomal protein genes and in the promoter regions of rRNA genes. Deletion of the gene encoding this protein affects expression of these and many other genes and impacts biofilm formation, secondary metabolite production, and fitness in fluctuating conditions. On the basis of these observations, we have designated the protein SutA (survival under transitions A).Pseudomonas aeruginosa | slow growth | transcription | proteomics | BONCAT T he cosmopolitan bacterium Pseudomonas aeruginosa is notorious as an opportunistic pathogen of burn wounds, medical devices, and the lungs of cystic fibrosis (CF) patients. The bacterium's genome is large and encodes an unusually high proportion of regulators (1). Compared with Escherichia coli, P. aeruginosa possesses more σ factors that direct RNA polymerase (RNAP) to promoter regions (24 vs. 7), more DNAbinding activators and repressors that enhance or prevent RNAP binding and transcription (∼550 vs. 150) (2, 3) and more small, noncoding RNAs (ncRNAs) that modulate the stability or translation of target transcripts (200 vs. 100) (4, 5). Much effort has been directed toward understanding the mechanisms by which this regulatory capacity governs the behaviors-such as quorum sensing, protein secretion, secondary metabolite production, and biofilm formation-that contribute to P. aeruginosa virulence.The physiological states of bacteria involved in chronic infections are substantially different from those most often studied in standard laboratory experiments; chronic infections are characterized by slow growth rates imposed by limited nutrients or oxidants or by host immune responses. Direct measurements of in situ microbial growth rates in the context of lung infections in CF patients have revealed doubling times of several days (6). Measurements of expectorated sputum show that hypoxic and anoxic zones exist within infected CF airways and can experience dramatic fluctuations in redox potential (7); P. aeruginosa strains isolated from the CF lung show gene expression patterns consistent with adaptations to hypoxia (8), suggesting ...

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“…BONCAT labeling, chemistry, and enrichment were performed as previously described (46). Label-free quantitation was used for the initial screen.…”

Section: Methodsmentioning

confidence: 99%

SutA is a bacterial transcription factor expressed during slow growth inPseudomonas aeruginosa

Babin

Bergkessel

Sweredoski

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Both studies used biorthogonal labeling of bacterial proteins with ANL to isolate the secretome ( Figure 3B). Mahdavi and coworkers [92] Only recently, ribosome profiling (or Ribo-seq) revolutionized the study of translation by deep sequencing of ribosome-protected mRNA fragments [95], thereby expanding our current understanding of the genomic architecture and coding potential for a wide variety of organisms to an unprecedented extent [96]. Moreover, recent ribosome profiling studies performed by us and others enabled translatome studies of bacterial cells, not only revealing translation of numerous previously unidentified (small) open reading frames, so-called pseudogenes and translation from numerous alternative translation initiation events including near-cognate start sites [97][98][99][100], but also empowering differential gene expression studies under infection relevant conditions [101].…”

Section: Secretomics For Identifying Bacterial Extracellular Effectormentioning

confidence: 99%

“…Recently however, enrichment of bacterial secretomes using in vitro infection models was reported [92,93]. Both studies used biorthogonal labeling of bacterial proteins with ANL to isolate the secretome ( Figure 3B).…”

Section: Secretomics For Identifying Bacterial Extracellular Effectormentioning

confidence: 99%

Omics in Aid of Host-pathogen Interaction Studies: A Bacterial Perspective

Fels¹,

Gevaert²,

Damme³

2017

Preprint

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By providing useful tools to study host-pathogen interactions, next-generation omics has recently enabled the study of gene expression changes in both pathogen and infected host simultaneously. However, since great discriminative power is required to study pathogen and host simultaneously throughout the infection process, the depth of quantitative gene expression profiling has proven to be unsatisfactory when focusing on bacterial pathogens, thus preferentially requiring specific strategies or the development of novel methodologies based on complementary omics approaches. In this review, we focus on the difficulties encountered when making use of omics approaches to study bacterial pathogenesis. Besides, we review different omics strategies (i.e. transcriptomics, proteomics and secretomics) and their applications for studying interactions of pathogens with their host.

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“…Being the first Yop effector protein to be translocated by the T3SS, 54 the 23 kDa YopE plays a major role in the initial bacterial defense against phagocytes. It does so by its GAP (GTPase activating protein) activity targeting the small Rho-GTPases Rac1, RhoG and partially also RhoA, thereby disrupting actin cytoskeleton dynamics ( Fig.…”

Section: Structure and Functionmentioning

confidence: 99%

Immunomodulatory Yersinia outer proteins (Yops)–useful tools for bacteria and humans alike

2017

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Human-pathogenic Yersinia produce plasmid-encoded Yersinia outer proteins (Yops), which are necessary to down-regulate anti-bacterial responses that constrict bacterial survival in the host. These Yops are effectively translocated directly from the bacterial into the target cell cytosol by the type III secretion system (T3SS). Cell-penetrating peptides (CPPs) in contrast are characterized by their ability to autonomously cross cell membranes and to transport cargoindependent of additional translocation systems. The recent discovery of bacterial cellpenetrating effector proteins (CPEs) -with the prototype being the T3SS effector protein YopM -established a new class of autonomously translocating immunomodulatory proteins. CPEs represent a vast source of potential self-delivering, anti-inflammatory therapeutics. In this review, we give an update on the characteristic features of the plasmid-encoded Yops and, based on recent findings, propose the further development of these proteins for potential therapeutic applications as natural or artificial cell-penetrating forms of Yops might be of value as bacteria-derived biologics.

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Identification of secreted bacterial proteins by noncanonical amino acid tagging

Cited by 83 publications

References 52 publications

SutA is a bacterial transcription factor expressed during slow growth inPseudomonas aeruginosa

SutA is a bacterial transcription factor expressed during slow growth inPseudomonas aeruginosa

Omics in Aid of Host-pathogen Interaction Studies: A Bacterial Perspective

Immunomodulatory Yersinia outer proteins (Yops)–useful tools for bacteria and humans alike

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