Ribosome reinitiation at leader peptides increases translation of bacterial proteins

Controlling the aggregation of vital bacterial proteins could be one of the new research directions and form the basis for the search and development of antibacterial drugs with targeted action. Such approach may be considered as an alternative one to antibiotics. Amyloidogenic regions can, like antibacterial peptides, interact with the “parent” protein, for example, ribosomal S1 protein (specific only for bacteria), and interfere with its functioning. The aim of the work was to search for peptides based on the ribosomal S1 protein from T. thermophilus, exhibiting both aggregation and antibacterial properties. The biological system of the response of Gram-negative bacteria T. thermophilus to the action of peptides was characterized. Among the seven studied peptides, designed based on the S1 protein sequence, the R23I (modified by the addition of HIV transcription factor fragment for bacterial cell penetration), R23T (modified), and V10I (unmodified) peptides have biological activity that inhibits the growth of T. thermophilus cells, that is, they have antimicrobial activity. But, only the R23I peptide had the most pronounced activity comparable with the commercial antibiotics. We have compared the proteome of peptide-treated and intact T. thermophilus cells. These important data indicate a decrease in the level of energy metabolism and anabolic processes, including the processes of biosynthesis of proteins and nucleic acids. Under the action of 20 and 50 μg/mL R23I, a decrease in the number of proteins in T. thermophilus cells was observed and S1 ribosomal protein was absent. The obtained results are important for understanding the mechanism of amyloidogenic peptides with antimicrobial activity and can be used to develop new and improved analogues.

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The Conservation of Low Complexity Regions in Bacterial Proteins Depends on the Pathogenicity of the Strain and Subcellular Location of the Protein

Genes ◽

10.3390/genes12030451 ◽

2021 ◽

Vol 12 (3) ◽

pp. 451

Author(s):

Pablo Mier ◽

Miguel A. Andrade-Navarro

Keyword(s):

Membrane Proteins ◽

Outer Membrane ◽

Bacterial Species ◽

Outer Membrane Proteins ◽

Subcellular Location ◽

Low Complexity ◽

Extracellular Proteins ◽

Bacterial Strains ◽

Bacterial Proteins ◽

Protein Subcellular Location

Low complexity regions (LCRs) in proteins are characterized by amino acid frequencies that differ from the average. These regions evolve faster and tend to be less conserved between homologs than globular domains. They are not common in bacteria, as compared to their prevalence in eukaryotes. Studying their conservation could help provide hypotheses about their function. To obtain the appropriate evolutionary focus for this rapidly evolving feature, here we study the conservation of LCRs in bacterial strains and compare their high variability to the closeness of the strains. For this, we selected 20 taxonomically diverse bacterial species and obtained the completely sequenced proteomes of two strains per species. We calculated all orthologous pairs for each of the 20 strain pairs. Per orthologous pair, we computed the conservation of two types of LCRs: compositionally biased regions (CBRs) and homorepeats (polyX). Our results show that, in bacteria, Q-rich CBRs are the most conserved, while A-rich CBRs and polyA are the most variable. LCRs have generally higher conservation when comparing pathogenic strains. However, this result depends on protein subcellular location: LCRs accumulate in extracellular and outer membrane proteins, with conservation increased in the extracellular proteins of pathogens, and decreased for polyX in the outer membrane proteins of pathogens. We conclude that these dependencies support the functional importance of LCRs in host–pathogen interactions.

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Genomic organization of the mouse granzyme A gene. Two mRNAs encode the same mature granzyme A with different leader peptides.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)74067-8 ◽

1992 ◽

Vol 267 (35) ◽

pp. 25488-25493

Author(s):

R.J. Hershberger ◽

H.K. Gershenfeld ◽

I.L. Weissman ◽

L Su

Keyword(s):

Genomic Organization ◽

Granzyme A ◽

Leader Peptides

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POS0384 A NOVEL MECHANISM LINKING MUCOSAL BACTERIA WITH AUTOANTIBODY RESPONSES IN RA: ACETYLATED BACTERIAL LYSATE AS A MODEL ANTIGEN

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2021-eular.1709 ◽

2021 ◽

Vol 80 (Suppl 1) ◽

pp. 422.1-422

Author(s):

M. Volkov ◽

A. S. B. Kampstra ◽

K. van Schie ◽

J. Kwekkeboom ◽

T. Huizinga ◽

...

Keyword(s):

B Cells ◽

Cell Activation ◽

Positive Control ◽

Negative Control ◽

B Cell Activation ◽

Human Fibrinogen ◽

Post Translational Modifications ◽

E Coli ◽

Bacterial Proteins ◽

Citrullinated Proteins

Background:Rheumatoid arthritis (RA) is characterized by autoantibodies against post-translationally modified proteins (AMPA) such as citrullinated, carbamylated and acetylated proteins. Importantly, these antibodies are highly multireactive, as they often recognize more than one of these post-translational modifications. Despite extensive research, the antigens inducing the breach of tolerance remain unknown, although microbial antigens are often suspected. Various bacteria are known to be capable of acetylation, therefore, it is intriguing to know what mechanisms can underlie the breach of tolerance towards acetylated proteins and development of anti-acetylated protein antibodies (AAPA).Objectives:To investigate whether acetylated proteins of bacterial origin (1) are recognized by human derived AMPA and AMPA expressing B cells; and (2) can induce AMPA development when used to immunize mice.Methods:Acetylated E. coli proteins were acquired with two separate methods (Figure 1A): by culturing E. coli in a condition promoting auto-acetylation (intrinsically acetylated bacterial proteins, IABP), or by directly acetylating lysate-derived proteins via a chemical reaction (extrinsically acetylated BP, EABP). Acetylated ovalbumin (AcOVA) served as positive control for AAPA induction in mice, non-acetylated BP (NABP) and phosphate buffer saline (PBS) served as negative control. Mice were immunized with these proteins and the resulting antibody response was studied by ELISA. Furthermore, EABP/IABP/NABP were investigated for recognition by human-derived AAPA with ELISA and AAPA-expressing B cells with spleen tyrosine kinase (Syk) phosphorylation assay; acetylated human fibrinogen and native fibrinogen served as positive and negative control.Results:Repetitive immunization of mice with EABP resulted in an AMPA response recognizing acetylated, carbamylated and citrullinated proteins. AMPA titers in these mice exceeded the titers in the positive control mice immunized with AcOVA and were substantially higher than in the NABP-immunized mice (Figure 1B). Human-derived monoclonal AAPA recognized EABP and IABP (not shown). B cell activation (measured by Syk phosphorylation) assay indicated that AAPA expressing B cells recognized EABP and (to a lesser extent) IABP, but not NABP (Figure 1C).Conclusion:Acetylated bacterial proteins are potent antigens that can induce cross-reactive AMPA responses in mice and they are recognized by human AAPA. This suggests that acetylated bacterial proteins could possibly be involved in the breach of tolerance in RA.Acknowledgements:We thank Dr. Can Araman and Prof. Chunaram Choudhary for their advice regarding optimization of bacterial auto-acetylation.Disclosure of Interests:None declared

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Dynamics ofMycobacterium tuberculosisAg85B Revealed by a Sensitive Enzyme-Linked Immunosorbent Assay

mBio ◽

10.1128/mbio.00611-19 ◽

2019 ◽

Vol 10 (2) ◽

Cited By ~ 2

Author(s):

Joel D. Ernst ◽

Amber Cornelius ◽

Miriam Bolz

Keyword(s):

Protein Secretion ◽

Immunosorbent Assay ◽

Bacterial Population ◽

Enzyme Linked Immunosorbent Assay ◽

Bacterial Protein ◽

Gram Positive ◽

Gram Negative ◽

Content Type ◽

Bacterial Proteins

ABSTRACTSecretion of specific proteins contributes to pathogenesis and immune responses in tuberculosis and other bacterial infections, yet the kinetics of protein secretion and fate of secreted proteinsin vivoare poorly understood. We generated new monoclonal antibodies that recognize theMycobacteriumtuberculosissecreted protein Ag85B and used them to establish and characterize a sensitive enzyme-linked immunosorbent assay (ELISA) to quantitate Ag85B in samples generatedin vitroandin vivo. We found that nutritional or culture conditions had little impact on the secretion of Ag85B and that there is considerable variation in Ag85B secretion by distinct strains in theM. tuberculosiscomplex: compared with the commonly used H37Rv strain (lineage 4),Mycobacteriumafricanum(lineage 6) secretes less Ag85B, and two strains from lineage 2 secrete more Ag85B. We also used the ELISA to determine that the rate of secretion of Ag85B is 10- to 100-fold lower than that of proteins secreted by Gram-negative and Gram-positive bacteria, respectively. ELISA quantitation of Ag85B in lung homogenates ofM. tuberculosisH37Rv-infected mice revealed that although Ag85B accumulates in the lungs as the bacterial population expands, the amount of Ag85B per bacterium decreases nearly 10,000-fold at later stages of infection, coincident with the development of T cell responses and arrest of bacterial population growth. These results indicate that bacterial protein secretionin vivois dynamic and regulated, and quantitation of secreted bacterial proteins can contribute to the understanding of pathogenesis and immunity in tuberculosis and other infections.IMPORTANCEBacterial protein secretion contributes to host-pathogen interactions, yet the process and consequences of bacterial protein secretion during infection are poorly understood. We developed a sensitive ELISA to quantitate a protein (termed Ag85B) secreted byM. tuberculosisand used it to find that Ag85B secretion occurs with slower kinetics than for proteins secreted by Gram-positive and Gram-negative bacteria and that accumulation of Ag85B in the lungs is markedly regulated as a function of the bacterial population density. Our results demonstrate that quantitation of bacterial proteins during infection can reveal novel insights into host-pathogen interactions.

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Glycosylation and biogenesis of a family of serine-rich bacterial adhesins

Microbiology ◽

10.1099/mic.0.025221-0 ◽

2009 ◽

Vol 155 (2) ◽

pp. 317-327 ◽

Cited By ~ 109

Author(s):

Meixian Zhou ◽

Hui Wu

Keyword(s):

Biosynthetic Pathway ◽

Protein Glycosylation ◽

Second Step ◽

Complex Nature ◽

Bacterial Physiology ◽

Bacterial Proteins ◽

New Family ◽

Bacterial Adhesins ◽

Platelet Binding ◽

Associated Proteins

Glycosylation of bacterial proteins is an important process for bacterial physiology and pathophysiology. Both O- and N-linked glycan moieties have been identified in bacterial glycoproteins. The N-linked glycosylation pathways are well established in Gram-negative bacteria. However, the O-linked glycosylation pathways are not well defined due to the complex nature of known O-linked glycoproteins in bacteria. In this review, we examine a new family of serine-rich O-linked glycoproteins which are represented by fimbriae-associated adhesin Fap1 of Streptococcus parasanguinis and human platelet-binding protein GspB of Streptococcus gordonii. This family of glycoproteins is conserved in streptococcal and staphylococcal species. A gene cluster coding for glycosyltransferases and accessory Sec proteins has been implicated in the protein glycosylation. A two-step glycosylation model is proposed. Two glycosyltransferases interact with each other and catalyse the first step of the protein glycosylation in the cytoplasm; the cross-talk between glycosylation-associated proteins and accessory Sec components mediates the second step of the protein glycosylation, an emerging mechanism for bacterial O-linked protein glycosylation. Dissecting the molecular mechanism of this conserved biosynthetic pathway offers opportunities to develop new therapeutic strategies targeting this previously unrecognized pathway, as serine-rich glycoproteins have been shown to play a role in bacterial pathogenesis.

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