Bacterial Protein Degradation Research Articles

Sequential application of cold plasma in chicken breast: Improvement of meat tenderness and bactericidal mechanism against histamine-producing Raoultella sp.

The feasibility of plasma-activated lactic acid solution (PALA) combined with dielectric barrier discharge (DBD) plasma treatment in chicken breast was evaluated. Results indicated that sequential plasma treatments slowed down the increase in the pH value and TVB-N value of meat during storage. Moreover, PALA treatment weakened the lipid oxidation triggered by DBD plasma treatment. Furthermore, centrifugal loss and shear force of chicken breast in the PALA-DBD group were reduced by 2.88% and 19.4%, respectively, and the myofibril fragmentation index was significantly increased compared to the control group, and PALA treatment increased free water content. Microbiological analyses showed that PALA-DBD treatment disrupted the cell membrane integrity of Raoultella sp., induced oxidative stress and reduced the activity of antioxidant enzymes (catalase and superoxide dismutase), led to the degradation of bacterial proteins and the disruption of α-helical structure.

BacPROTACs mediate targeted protein degradation in bacteria

SummaryHijacking the cellular protein degradation system offers unique opportunities for drug discovery, as exemplified by proteolysis-targeting chimeras. Despite their great promise for medical chemistry, so far, it has not been possible to reprogram the bacterial degradation machinery to interfere with microbial infections. Here, we develop small-molecule degraders, so-called BacPROTACs, that bind to the substrate receptor of the ClpC:ClpP protease, priming neo-substrates for degradation. In addition to their targeting function, BacPROTACs activate ClpC, transforming the resting unfoldase into its functional state. The induced higher-order oligomer was visualized by cryo-EM analysis, providing a structural snapshot of activated ClpC unfolding a protein substrate. Finally, drug susceptibility and degradation assays performed in mycobacteria demonstrate in vivo activity of BacPROTACs, allowing selective targeting of endogenous proteins via fusion to an established degron. In addition to guiding antibiotic discovery, the BacPROTAC technology presents a versatile research tool enabling the inducible degradation of bacterial proteins.

Protein degradation control and regulation of bacterial survival and pathogenicity: the role of protein degradation systems in bacteria.

Protein degradation systems play crucial roles in all the kingdoms of life. Their natural function is to eliminate proteins that are improperly synthesized, damaged, aggregated, or short-lived, ensuring the timely and accurate regulation of the response to abrupt environmental changes. Thus, proteolysis plays an important role in protein homeostasis, quality control, and the control of regulatory processes, such as adaptation and cell development. Except for the lysosome, ATPases Associated with various cellular Activities (AAA+) ATPase-protease complex is another major protein degradation system in the cell. The AAA+ ATPase-protease complex is a giant energy-dependent protease complex found in almost all kinds of cells, including bacteria, archaea and eukarya. Based on sequence analysis of ClpQ (HslV) and 20S proteasome beta subunits, it was found that bacterial ClpQ possess multiple same highly conserved motifs with 20S proteasome beta subunits of archaea and eukaryote. In this review, we also discussed the structure and functional mechanism, protein degradation signals and pathogenic role of proteasome / Clp protease complex in prokaryotes. Bacterial protein degradation systems play important roles in stress tolerance, protein quality control, DNA protection, transcription and pathogenicity of bacteria. But our current knowledge of the bacterial protease system is incomplete, and further research into the Clp protease complex and associated protein degradation signals will extend our understanding of the metabolism, physiology, reproduction, and pathogenicity of bacteria.

Cargo competition for a dimerization interface restricts and stabilizes a bacterial protease adaptor

Bacterial protein degradation is a regulated process aided by protease adaptors that alter specificity of energy-dependent proteases. In Caulobacter crescentus, cell cycle-dependent protein degradation depends on a hierarchy of adaptors, such as the dimeric RcdA adaptor, which binds multiple cargo and delivers substrates to the ClpXP protease. RcdA itself is degraded in the absence of cargo, and how RcdA recognizes its targets is unknown. Here, we show that RcdA dimerization and cargo binding compete for a common interface. Cargo binding separates RcdA dimers, and a monomeric variant of RcdA fails to be degraded, suggesting that RcdA degradation is a result of self-delivery. Based on HDX-MS studies showing that different cargo rely on different regions of the dimerization interface, we generate RcdA variants that are selective for specific cargo and show cellular defects consistent with changes in selectivity. Finally, we show that masking of cargo binding by dimerization also limits substrate delivery to restrain overly prolific degradation. Using the same interface for dimerization and cargo binding offers an ability to limit excess protease adaptors by self-degradation while providing a capacity for binding a range of substrates.

Cargo binding of an adaptor directly competes with oligomerization

Bacterial protein degradation is a tightly coordinated process aided by protease adaptors that coordinate cell cycle and manages stress responses. In Caulobacter crescentus, cell‐cycle dependent protein degradation depends on the highly conserved ClpXP protease and several ClpXP specific adaptors that dictate hierarchal substrate degradation. While most protease adaptors deliver only a single substrate, the RcdA adaptor directly binds and coordinates the degradation of several cell‐cycle regulated substrates that share no obvious sequence or structural similarities. Here we show that cargo binding uses the same interface as that responsible for dimerization of RcdA. Cargo binding converts RcdA from a homodimer to a monomer and this effect extends to all RcdA cargo. We identify key residues in this interface with hydrogen‐deuterium exchange mass spectrometry and make mutations to generate a monomeric variant incapable of dimerization that also fails to bind cargo. Interestingly, RcdA itself is normally degraded by ClpXP unless bound to cargo, but the monomeric variant fails to be degraded, suggesting that RcdA delivers cargo or delivers itself to ClpXP. Expressing a monomeric RcdA in vivo results in irregular stalk formation and loss of normal cargo turnover. Our work shows how cargo binding competes with self‐dimerization of RcdA through direct competition for a common interface and that disruption of this interface results in defects in normal cell physiology.

Xenogeneic modulation of the ClpCP protease of Bacillus subtilis by a phage-encoded adaptor-like protein

Like eukaryotic and archaeal viruses, which coopt the host's cellular pathways for their replication, bacteriophages have evolved strategies to alter the metabolism of their bacterial host. SPO1 bacteriophage infection of Bacillus subtilis results in comprehensive remodeling of cellular processes, leading to conversion of the bacterial cell into a factory for phage progeny production. A cluster of 26 genes in the SPO1 genome, called the host takeover module, encodes for potentially cytotoxic proteins that specifically shut down various processes in the bacterial host, including transcription, DNA synthesis, and cell division. However, the properties and bacterial targets of many genes of the SPO1 host takeover module remain elusive. Through a systematic analysis of gene products encoded by the SPO1 host takeover module, here we identified eight gene products that attenuated B. subtilis growth. Of the eight phage gene products that attenuated bacterial growth, a 25-kDa protein called Gp53 was shown to interact with the AAA+ chaperone protein ClpC of the ClpCP protease of B. subtilis. Our results further reveal that Gp53 is a phage-encoded adaptor-like protein that modulates the activity of the ClpCP protease to enable efficient SPO1 phage progeny development. In summary, our findings indicate that the bacterial ClpCP protease is the target of xenogeneic (dys)regulation by a SPO1 phage–derived factor and add Gp53 to the list of antibacterial products that target bacterial protein degradation and therefore may have utility for the development of novel antibacterial agents.

The Mycobacterium tuberculosis Pup-proteasome system regulates nitrate metabolism through an essential protein quality control pathway

The human pathogen Mycobacterium tuberculosis encodes a proteasome that carries out regulated degradation of bacterial proteins. It has been proposed that the proteasome contributes to nitrogen metabolism in M. tuberculosis, although this hypothesis had not been tested. Upon assessing M. tuberculosis growth in several nitrogen sources, we found that a mutant strain lacking the Mycobacterium proteasomal activator Mpa was unable to use nitrate as a sole nitrogen source due to a specific failure in the pathway of nitrate reduction to ammonium. We found that the robust activity of the nitrite reductase complex NirBD depended on expression of the groEL/groES chaperonin genes, which are regulated by the repressor HrcA. We identified HrcA as a likely proteasome substrate, and propose that the degradation of HrcA is required for the full expression of chaperonin genes. Furthermore, our data suggest that degradation of HrcA, along with numerous other proteasome substrates, is enhanced during growth in nitrate to facilitate the derepression of the chaperonin genes. Importantly, growth in nitrate is an example of a specific condition that reduces the steady-state levels of numerous proteasome substrates in M. tuberculosis.

Proteasome Accessory Factor C (pafC) Is a novel gene Involved in Mycobacterium Intrinsic Resistance to broad-spectrum antibiotics--Fluoroquinolones.

Antibiotics resistance poses catastrophic threat to global public health. Novel insights into the underlying mechanisms of action will inspire better measures to control drug resistance. Fluoroquinolones are potent and widely prescribed broad-spectrum antibiotics. Bacterial protein degradation pathways represent novel druggable target for the development of new classes of antibiotics. Mycobacteria proteasome accessory factor C (pafC), a component of bacterial proteasome, is involved in fluoroquinolones resistance. PafC deletion mutants are hypersensitive to fluoroquinolones, including moxifloxacin, norfloxacin, ofloxacin, ciprofloxacin, but not to other antibiotics such as isoniazid, rifampicin, spectinomycin, chloramphenicol, capreomycin. This phenotype can be restored by complementation. The pafC mutant is hypersensitive to H2O2 exposure. The iron chelator (bipyridyl) and a hydroxyl radical scavenger (thiourea) can abolish the difference. The finding that pafC is a novel intrinsic selective resistance gene provided new evidence for the bacterial protein degradation pathway as druggable target for the development of new class of antibiotics.

Rapid and tunable post-translational coupling of genetic circuits

One promise of synthetic biology is the creation of genetic circuitry that enables the execution of logical programming in living cells. Such “wet programming” is positioned to transform a wide and diverse swath of biotechnology ranging from therapeutics and diagnostics to water treatment strategies. While progress in the development of a library of genetic modules continues apace1–4, a major challenge for their integration into larger circuits is the generation of sufficiently fast and precise communication between modules5,6. An attractive approach is to integrate engineered circuits with host processes that facilitate robust cellular signaling7. In this context, recent studies have demonstrated that bacterial protein degradation can trigger a precise response to stress by overloading a limited supply of intracellular proteases8–10. Here, we use protease competition to engineer rapid and tunable coupling of genetic circuits across multiple spatial and temporal scales. We characterize coupling delay times that are more than an order of magnitude faster than standard transcription-factor based coupling methods (less than one minute compared with ~20–40 minutes) and demonstrate tunability through manipulation of the linker between the protein and its degradation tag. We use this mechanism as a platform to couple genetic clocks at the intracellular and colony level, then synchronize the multi-colony dynamics to reduce variability in both clocks. We show how the coupled clock network can be used to encode independent environmental inputs into a single time series output, thus enabling the possibility of frequency multiplexing in a genetic circuit context. Our results establish a general framework for the rapid and tunable coupling of genetic circuits through the use of native queueing processes such as protein degradation.

A Multifaceted Study of Pseudomonas aeruginosa Shutdown by Virulent Podovirus LUZ19

ABSTRACTIn contrast to the rapidly increasing knowledge on genome content and diversity of bacterial viruses, insights in intracellular phage development and its impact on bacterial physiology are very limited. We present a multifaceted study combining quantitative PCR (qPCR), microarray, RNA-seq, and two-dimensional gel electrophoresis (2D-GE), to obtain a global overview of alterations in DNA, RNA, and protein content in Pseudomonas aeruginosa PAO1 cells upon infection with the strictly lytic phage LUZ19. Viral genome replication occurs in the second half of the phage infection cycle and coincides with degradation of the bacterial genome. At the RNA level, there is a sharp increase in viral mRNAs from 23 to 60% of all transcripts after 5 and 15 min of infection, respectively. Although microarray analysis revealed a complex pattern of bacterial up- and downregulated genes, the accumulation of viral mRNA clearly coincides with a general breakdown of abundant bacterial transcripts. Two-dimensional gel electrophoretic analyses shows no bacterial protein degradation during phage infection, and seven stress-related bacterial proteins appear. Moreover, the two most abundantly expressed early and late-early phage proteins, LUZ19 gene product 13 (Gp13) and Gp21, completely inhibit P. aeruginosa growth when expressed from a single-copy plasmid. Since Gp13 encodes a predicted GNAT acetyltransferase, this observation points at a crucial but yet unexplored level of posttranslational viral control during infection.

Bacterial protein degradation by different rumen protozoal groups1

Bacterial predation by protozoa has the most deleterious effect on the efficiency of N use within the rumen, but differences in activity among protozoal groups are not completely understood. Two in vitro experiments were conducted to identify the protozoal groups more closely related with rumen N metabolism. Rumen protozoa were harvested from cattle and 7 protozoal fractions were generated immediately after sampling by filtration through different nylon meshes at 39 °C, under a CO(2) atmosphere to maintain their activity. Protozoa were incubated with (14)C-labeled bacteria to determine their bacterial breakdown capacity, according to the amount of acid-soluble radioactivity released. Epidinium tended to codistribute with Isotricha and Entodinium with Dasytricha; therefore, their activity was calculated together. This study demonstrated that big Diplodiniinae had the greatest activity per cell (100 ng bacterial CP per protozoa and hour), followed by Epidinium plus Isotricha (36.4), small Diplodiniinae (34.2), and Entodinium plus Dasytricha (14.8), respectively. However, the activity per unit of protozoal volume seemed to vary, depending on the protozoal taxonomy. Small Diplodiniinae had the greatest activity per volume (325 ng bacterial CP per protozoal mm(3) and hour), followed by big Diplodiniinae (154), Entodinium plus Dasytricha (104), and Entodinium plus Dasytricha (25.6). A second experiment was conducted using rumen fluid from holotrich-monofaunated sheep. This showed that holotrich protozoa had a limited bacterial breakdown capacity per cell (Isotricha 9.44 and Dasytricha 5.81 ng bacterial CP per protozoa and hour) and per protozoal volume (5.97 and 76.9 ng bacterial CP per protozoal mm(3) and hour, respectively). Therefore, our findings indicated that a typical protozoal population (10(6) total protozoa/mL composed by Entodinium sp. 88%, Epidinium sp. 7%, and other species 4%) is able to break down ~17% of available rumen bacteria every hour. Entodinium sp. is responsible for most of this bacterial breakdown (70 to 75%), followed by Epidinium sp. (16 to 24%), big Diplodiniinae (4 to 6%), and small Diplodiniinae (2 to 6%), whereas holotrich protozoa have a negligible activity (Dasytricha sp. 0.6 to 1.2% and Isotricha sp. 0.2 to 0.5%). This in vitro information must be carefully interpreted, but it can be used to indicate which protozoal groups should be suppressed to improve microbial protein synthesis in vivo.

Photodynamic Inactivation of Methylene Blue and Tungsten‐Halogen Lamp Light against Food Pathogen Listeria monocytogenes

The aim of this study was to verify the bactericidal effect and the damage of photodynamic inactivation (PDI) using methylene blue (MB) and tungsten-halogen lamp over Listeria monocytogenes via atomic force microscopy, absorption spectrophotometry, agarose gel electrophoresis, real-time PCR and SDS-PAGE. The obtained data indicated that the viability of L. monocytogenes was ca 7-log reduced by illumination with 10 min tungsten-halogen lamp light under the presence of 0.5 μg mL(-1) MB, and this bactericidal activity against L. monocytogenes of PDI increased proportionally to the concentration of MB and the duration of irradiation. Moreover, after irradiation with MB and visible light, the leakage of intracellular contents was estimated by spectrophotometer at OD(260) and OD(280), which correlated with morphological alterations. Furthermore, genomic DNA cleavage and protein degradation were also detected after PDI treatment. Consequently, breakage of the membrane, damage of the genomic DNA and degradation of bacterial proteins may play an important role in the mechanisms involved in PDI-MB bactericidal activity on L. monocytogenes.

Support for a potential role of E. coli oligopeptidase A in protein degradation

Protein degradation is an essential quality control and regulatory function in organisms ranging from bacteria to eukaryotes. In bacteria, this process is initiated by ATP-dependent proteases which digest proteins to short peptides that are subsequently hydrolyzed to smaller fragments and free amino acids. While the entire genome of Escherichia coli has been sequenced, identification of endopeptidases that perform this downstream hydrolysis remains incomplete. However, in eukaryotes, thimet oligopeptidases (TOP) has been shown to hydrolyze peptides generated by the degradation of proteins by the 26S proteasome. These findings motivated us to investigate whether E. coli oligopeptidase A (OpdA), a homolog of TOP might play a similar general role in bacterial protein degradation. Herein, we provide initial support for this hypothesis by demonstrating that OpdA efficiently cleaves the peptides generated by the activity of the three primary ATP-dependent proteases from E. coli—Lon, HslUV, and ClpAP.

A nonamer peptide derived from Listeria monocytogenes metalloprotease is presented to cytolytic T lymphocytes.

Listeria monocytogenes is an intracellular bacterium that secretes proteins into the cytosol of infected macrophages. Major histocompatibility complex (MHC) class I molecules bind peptides that are generated by the degradation of bacterial proteins and present them to cytolytic T lymphocytes (CTL). In this study we have investigated CTL responses in L. monocytogenes-immunized mice to peptides that (i) derive from the L. monocytogenes proteins phosphatidylinositol-specific phospholipase C, lecithinase (most active on phosphatidylcholine), metalloprotease (Mpl), PrfA, and the ORF-A product and (ii) conform to the binding motif of the H2-Kd MHC class I molecule. We identified a nonamer peptide, Mpl 84-92, that is presented to L. monocytogenes-specific CTL by H2-Kd MHC class I molecules. Unlike other motif-conforming peptides derived from the secreted Mpl of L. monocytogenes, Mpl 84-92 is bound with high affinity by H2-Kd. Mpl 84-92 is the fourth L. monocytogenes-derived peptide found to be presented to CTL by the H2-Kd molecule during infection and demonstrates the importance of high-affinity interactions between antigenic peptides and MHC class I molecules for CTL priming.

Immunity and mycobacterial infection : new generation vaccines for leprosy

One of the greatest triumphs of medical science has been the development of vaccines that provide protective immunity from infectious diseases. These vaccines have been based on the isolation of the infectious agent and the subsequent inactivation or attenuation of the agent for use in immunization to yield immune responses of a protective nature. There are a number of diseases where this approach has led to immune responses but not to the development of protective immunity. These include a range of tropical diseases caused by protozoan parasites. leprosy, tuberculosis and AIDS. In developing a vaccine for leprosy there appear to be three relevant arguments to consider. First, most individuals who are infected with Mycobacterium leprae develop protective immunity and do not get clinical disease. Second, it is apparent that the clinical spectrum of disease can be correlated with cell-mediated immune responses to M.leprae. Third, while these observations are indicative that, in susceptible individuals, the immune response to M.leprae has a dramatic effect on the clinical spectrum of the disease, they do not throw light on the immunological difference between individuals that develop leprosy and those who are infected but develop protective immunity. Analyses of recombinant proteins isolated from genomic libraries of M.leprae have yielded chemically-defined new immunologically-reactive epitopes. These epitopes are peptides which are produced by degradation of bacterial proteins in antigen-presenting cells. The peptides which represent the epitopes associate with newly synthesized class II molecules of the major histocompatibility complex. I will describe our analysis of the 18kD protein antigen of Mycobacterium leprae and discuss ways that peptide fragments may be engineered into new vaccines and presented to individuals for immunization. The immunological basis of protective immunity will be reviewed. One of the greatest triumphs of medical science has been the development of vaccines that provide protective immunity from infectious diseases. These vaccines have been based on the isolation of the infectious agent and the subsequent inactivation or attenuation of the agent for use in immunization to yield immune responses of a protective nature. There are a number of diseases where this approach has led to immune responses but not to the development of protective immunity. These include a range of tropical diseases caused by protozoan parasites. leprosy, tuberculosis and AIDS. In developing a vaccine for leprosy there appear to be three relevant arguments to consider. First, most individuals who are infected with Mycobacterium leprae develop protective immunity and do not get clinical disease. Second, it is apparent that the clinical spectrum of disease can be correlated with cell-mediated immune responses to M.leprae. Third, while these observations are indicative that, in susceptible individuals, the immune response to M.leprae has a dramatic effect on the clinical spectrum of the disease, they do not throw light on the immunological difference between individuals that develop leprosy and those who are infected but develop protective immunity. Analyses of recombinant proteins isolated from genomic libraries of M.leprae have yielded chemically-defined new immunologically-reactive epitopes. These epitopes are peptides which are produced by degradation of bacterial proteins in antigen-presenting cells. The peptides which represent the epitopes associate with newly synthesized class II molecules of the major histocompatibility complex. I will describe our analysis of the 18kD protein antigen of Mycobacterium leprae and discuss ways that peptide fragments may be engineered into new vaccines and presented to individuals for immunization. The immunological basis of protective immunity will be reviewed.

Respiratory burst facilitates the digestion of Escherichia coli killed by polymorphonuclear leukocytes.

We examined factors that may limit degradation of bacterial protein of Escherichia coli S15 killed by polymorphonuclear leukocytes (PMN). Both human and rabbit PMN degraded up to 40% of [14C]amino acid-labeled protein of ingested and killed E. coli in 2 h as determined by loss of acid-precipitable radioactivity. In contrast, equally bactericidal broken-PMN preparations or isolated granules degraded only about 10% of bacterial protein regardless of pH. To determine whether activation of the respiratory burst contributes to digestion, we compared degradation by intact PMN in room air and under N2. Depletion of O2 by N2 flushing had no effect on the bactericidal activity of either human or rabbit PMN but reduced degradation by approximately 50%. Protein degradation during phagocytosis was also reduced in the presence of cyanide or azide, inhibitors of myeloperoxidase (MPO). PMN of two patients with chronic granulomatous disease ingested and killed E. coli S15 as well as did normal PMN but degraded bacterial protein as did normal PMN incubated under N2. The low degradative activity of PMN disrupted by sonication could be raised to nearly the level of intact PMN incubated in room air by preincubation of the PMN with 10(-7) M formyl-methionyl-leucyl-phenylalanine (fMLP) before sonication and by pretreatment of E. coli with MPO. Depletion of O2 or chloride during these preincubations with formyl-methionyl-leucyl-phenylalanine respectively, virtually abolished and markedly diminished stimulation of bacterial protein degradation. We conclude that enhanced MPO-mediated O2 metabolism of intact PMN plays a role in the digestion of killed E. coli.

Correlation between rates of degradation of bacterial proteins in vivo and their sensitivity to proteases.

Experiments were undertaken to test whether the relative rates of degradation of proteins in vivo might correlate with their sensitivity to proteases. Various experimental conditions that promote degradation of proteins in Escherichia coli increased sensitivity of average cell proteins to trypsin or other endoproteases, including incorporation of several aminoacid analogs into the proteins, incorporation of puromycin into the polypeptide chain, or frequent errors in translation. Those abnormal proteins that were degraded most rapidly within the cell appear responsible for the increased protease sensitivity of the cell extract. Normal E. coli proteins that rapidly turn over in growing cells are, on the average, more sensitive to trypsin or Pronase than cell proteins that turn over more slowly. The inherent sensitivity of a protein to proteolytic digestion is thus a major determinant of protein half-lives in vivo.