Gram positive protein secretion

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Gram positive protein secretion

• Nikhil Thomas
• Microbiology and Immunology
• n.thomas_at_dal.ca

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Relevance in human disease

• MRSA
• Methicillin resistant Staphylococcus aureus
• C. difficile
• Group A streptococci (GAS)
• Flesh eating disease
• Vancomycin resistant Enterococci
• VRE
• Streptococcus pnemoniae
• Bacillus anthracis (Anthrax)

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Features of the Gram cell

• Relatively simple architecture compared to Gram
  negatives
• Cytoplasm
• Cellular membrane
• Cell wall
• Linked techoic acids, peptidoglycan
• Can have capsule (EPS)
• Surface layer (S-layers)
• These extras comprise the cell envelope

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Gramve protein secretion

• Secreted proteins often have
• Signal peptide at the amino terminal region
• 15-20 hydrophobic aa stretch flanked by
  positively charged residues
• Proteins are secreted using the Sec system plus
  any auxiliary or associated proteins in the
  cytoplasm
• Many more signal peptidases are found in gram
  ves (7) compared to gram negs (2)

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Anchoring of secreted proteins

• Achieved using sortase enzymes
• Sortase enzymes are membrane localized via signal
  peptides
• Function as a transpeptidase
• Forms an acyl intermediate with the cleaved
  surface protein
• Links protein to penta-glycine cross-bridge of PG
• Active site cysteine residue
• Sortase mediates the covalent linkage of proteins
  to the cell wall
• Important for bacterial cell survival during
  infection

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The sortase linkage reaction steps
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Structure of Sortase A bound to a peptide
substrate
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Examples of anchored proteins

• Fibronectin binding proteins
• Collagen adhesion protein (Cna)
• Iron binding proteins
• Critical for survival where iron is limited (low
  solubility)
• Cell surface pilins (in the pili)
• Protein A
• Binds to Fc terminus of human immunoglobulins in
  a non-immune fashion

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• Acquisition of heme- iron from the bloodstream by
  gram positive bacteria

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Sortase mediates anchoring of proteins to the
cell envelope
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Sorting (out) signals

• A C-terminal cell sorting signal within the
  protein
• 35aa stretch encompassing a conserved LPXTG
  motif (X is any amino acid)
• Deletion of this motif abrogates sorting and cell
  wall linkage
• In the case of protein A no secretion either
• Sorting signals can be anywhere in the protein
  but require an upstream signal peptide

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Biogenesis of pili

• Corynebacterium diptherium
• Upper respiratory tract infection
• Diptheria toxin (tissue damage)
• Pili are likely involved in adherence to the
  respiratory tract
• Promote colonization, followed by infection,
  tissue damage, disease.

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Immunostaining for localization studies in situ
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Formation of pili mediated by sortase
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Review

• Know the difference aspects of type I-IV
  secretion, Sec system.
• Specifics are important (need to know)
• Think in terms of distictions
• Gram positive and Gram negative barriers to
  transverse
• Recall the functional definition secretion
• Questions in the form of facts but also in the
  context of problem solving

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Literature

• Marraffini et al., 2006 MMBR 192-221
• Sorting and the Art of Anchoring Proteins to the
  Envelopes of Gram positive Bacteria
• Papanikou et al., 2007 NRM 5 839-851
• Bacterial protein secretion through the
  translocase nanomachine
• Delepelaire P. 2004 Biochimica et Biophysica Acta
  1694 149 161
• Type I secretion in gram-negative bacteria
• Ghosh P. 2004 MMBR Dec. 2004 771-795
• Process of Protein Transport by the Type III
  Secretion System
• Christie PJ 2003 NRM 1 137-149
• THE VERSATILE BACTERIAL TYPE IV SECRETION SYSTEMS