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Cited by 73Antimicrobial Resistance in StaphylococcusMicrobial infections and disease researchBacterial Genetics and Biotechnology

Staphylococcus aureus protein A mediates invasion across airway epithelial cells through activation of RhoA GTPase signaling and proteolytic activity

Open access
Soong, Grace, Martin, Francis J., Chun, Jarin, Cohen, Taylor S., Ahn, Danielle S., Prince, Alice·2011·Journal of Biological Chemistry
DOI 10.1074/jbc.m111.295386PMC PMC3195615
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Abstract

Staphyococcus aureus and especially the epidemic methicillin-resistant S. aureus strains cause severe necrotizing pneumonia. The mechanisms whereby these organisms invade across the mucosal epithelial barrier to initiate invasive infection are not well understood. Protein A (SpA), a highly conserved and abundant surface protein of S. aureus, activates TNF receptor 1 and EGF receptor (EGFR) signaling cascades that can perturb the cytoskeleton. We demonstrate that wild-type S. aureus, but not spa mutants, invade across polarized airway epithelial cell monolayers via the paracellular junctions. SpA stimulated a RhoA/ROCK/MLC cascade, resulting in the contraction of the cytoskeleton. SpA(+) but not SpA(-) mutants stimulated activation of EGFR and along with subsequent calpain activity cleaved the membrane-spanning junctional proteins occludin and E-cadherin, facilitating staphylococcal transmigration through the cell-cell junctions. Treatment of polarized human airway epithelial monolayers with inhibitors of ROCK, EGFR, MAPKs, or calpain prevented staphylococcal penetration through the monolayers. In vivo, blocking calpain activity impeded bacterial invasion into the lung parenchyma. Thus, S. aureus exploits multiple receptors available on the airway mucosal surface to facilitate invasion across epithelial barriers.

AI snapshot

Written from the paper’s full text.

TL;DR

S. aureus protein A (SpA) is required for staphylococcal transmigration across polarized airway epithelial monolayers via paracellular junctions, activating RhoA/ROCK/MLC signaling and EGFR/calpain-dependent cleavage of junctional proteins. SpA alone is not sufficient; intact bacteria and additional signals are needed, and in vivo calpain inhibition reduced invasion while ROCK inhibition had harmful off-target effects.

Key findings

  • Wild-type S. aureus Newman crossed polarized 16HBE monolayers, with approximately 1 × 10^6 cfu/ml recovered basolaterally from a 1 × 10^9 cfu/ml apical inoculum; neither spa nor sortase mutants crossed, and exogenous purified SpA restored transmigration.
  • SpA+ bacteria activated RhoA, ROCK, and MLC phosphorylation; the ROCK inhibitor Y-27632 caused a dose-dependent decrease in staphylococcal transmigration.
  • SpA+ but not SpA− bacteria stimulated EGFR/MAPK and calpain activity, generating occludin and E-cadherin cleavage products; inhibitors of EGFR, ERK1/2, JNK, p38, or calpain blocked transmigration.
  • In vivo, calpeptin pretreatment significantly increased staphylococci in bronchoalveolar lavage fluid (p = 0.0043), suggesting impaired invasion; gefitinib did not inhibit USA300 invasion despite reducing ERK1/2 phosphorylation; ROCK inhibition increased mortality (p = 0.0095) and blocked PMN mobility.

Methods

  • Polarized human 16HBE airway epithelial cells grown on 3-µm Transwell filters at an air-liquid interface; transmigration assays used S. aureus Newman wild type, spa mutant, sortase mutant, and LAC USA300 MRSA, with purified SpA, TNF, TGFα, and signaling inhibitors.
  • Signaling and proteolysis readouts included Rho activation G-LISA and Rhotekin RBD-agarose immunoblot, Western blotting for phospho-MLC and occludin/E-cadherin cleavage, confocal microscopy, and dextran permeability.
  • Mouse pneumonia model used 8-week-old female C57BL/6 mice inoculated intranasally with 10^8 cfu S. aureus or PBS; pretreatments included Y-27632, gefitinib, and calpeptin; BAL/lung CFU and PMN flow cytometry were assessed, with t test, ANOVA/Dunnett, and Mann-Whitney tests (p < 0.05).

Limitations

  • No explicit limitations section is present. The text notes that mice have a general lack of susceptibility to S. aureus infection, raising doubts about the applicability of mouse data to human infection.
  • In vivo ROCK inhibition was confounded by blocking PMN RhoA signaling and mobility and causing increased morbidity and mortality, limiting interpretation of ROCK’s in vivo role.
  • SpA was necessary but not sufficient: purified SpA alone did not alter junctions or dextran permeability, and TNF or TGFα did not complement the spa mutant, implicating additional intact-bacteria signals such as lipoproteins/TLR2; in vivo EGFR inhibition also failed to inhibit invasion despite target engagement.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1074/jbc.m111.295386

Cites

43 of 43 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 8 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    Pretreatment with calpeptin resulted in significantly increased numbers of staphylococci in bronchoalveolar lavage fluid compared with vehicle-treated controls.

    “Pretreatment with calpeptin resulted in significantly increased numbers of staphylococci in the bronchoalveolar lavage fluid compared with vehicle-treated controls (Fig. 6D), suggesting that inhibition of calpains interfered with staphylococcal invasion across the airway epithelium.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The mouse model of S. aureus pneumonia is applicable to human invasive pulmonary infection.
  • Findingown data

    In vivo, blocking calpain activity impeded bacterial invasion into the lung parenchyma.

    “In vivo, blocking calpain activity impeded bacterial invasion into the lung parenchyma.”
    • Also stated in: § EGFR-induced Proteolytic Activity · hedged
      “Pretreatment with calpeptin resulted in significantly increased numbers of staphylococci in the bronchoalveolar lavage fluid compared with vehicle-treated controls (Fig. 6D), suggesting that inhibition of calpains interfered with staphylococcal invasion across the airway epithelium.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The mouse model of S. aureus pneumonia is applicable to human invasive pulmonary infection.
  • Findingown data

    Wild-type S. aureus, but not spa mutants, invade across polarized airway epithelial cell monolayers via the paracellular junctions.

    “We demonstrate that wild-type S. aureus, but not spa mutants, invade across polarized airway epithelial cell monolayers via the paracellular junctions.”
    • Also stated in: § SpA Alters the Barrier Properties of Epithelial Monolayers- · plain
      “Approximately 1 ϫ 10 6 cfu/ml of a 1 ϫ 10 9 cfu/ml inoculum of strain Newman were quantified in the basal compartment; whereas neither the spa nor sortase mutants crossed the monolayer during this period of time.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Transmigration across polarized 16HBE airway epithelial monolayers is a valid model of S. aureus invasion across the human airway epithelium.
  • Findingown data

    Purified SpA alone did not alter the junctions.

    “Of note, treatment of the monolayers with purified SpA alone did not alter the junctions as detected by monitoring the transmigration of fluorescent dextran across monolayers (Fig. 4C) or by visualizing the distribution of ZO-1 and actin (Fig. 4D).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Transmigration across polarized 16HBE airway epithelial monolayers is a valid model of S. aureus invasion across the human airway epithelium.
  • Findingown data

    Wild-type S. aureus, but not a spa mutant, induced active Rho GTP detected by immunoblotting.

    “Wild-type S. aureus, but not a spa mutant, induced active Rho GTP detected by immunoblotting (Fig. 3B).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Transmigration across polarized 16HBE airway epithelial monolayers is a valid model of S. aureus invasion across the human airway epithelium.
  • May combine two claims. 16
  • Classified as probably not a substantial claim. 1

  • May combine two claims.

    Findingown data

    Treatment with calpain inhibitor calpeptin significantly blocked staphylococcal penetration through epithelial monolayers, whereas GM6001 had no effect and TAPI had only a modest effect.

    “Treatment of the monolayers with the general calpain inhibitor calpeptin significantly blocked staphylococcal penetration through the epithelial monolayers (Fig. 6A), whereas a general protease inhibitor (GM6001), had no effect, and TAPI, which is specific for ADAM17, had only a modest effect on S. aureus transmigration.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Transmigration across polarized 16HBE airway epithelial monolayers is a valid model of S. aureus invasion across the human airway epithelium.

  • May combine two claims.

    Findingown data

    TNF did not complement the transmigration defect of the spa mutant, nor did TGFα or both cytokines together.

    “However, the addition of TNF did not complement the transmigration defect of the spa mutant, nor did the addition of TGF␣, an EGFR agonist, or both cytokines together (Fig. 4A).”
    • Also stated in: § DISCUSSION · plain
      “TNF did not activate significant changes in the cytoskeleton of human respiratory epithelial cells, nor did TNF complement a spa mutant.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Transmigration across polarized 16HBE airway epithelial monolayers is a valid model of S. aureus invasion across the human airway epithelium.

  • May combine two claims.

    Findingown data

    Systemic treatment of mice with Y-27632 blocked PMN RhoA signaling, inhibiting their mobility and causing significantly increased morbidity and mortality.

    “systemic treatment of mice with Y-27632 also blocked PMN RhoA signaling in PMNs, inhibiting their mobility (Fig. 3F) and causing significantly increased morbidity and mortality (Fig. 3G).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The mouse model of S. aureus pneumonia is applicable to human invasive pulmonary infection.

  • May combine two claims.

    Findingown data

    MRSA (LAC USA300) penetrate through paracellular junctions and traverse monolayers, whereas spa mutant remains at the apical surface.

    “Using biotin as a marker for exposed proteins, MRSA are observed to penetrate through the paracellular junctions and traverse the monolayers in contrast to the spa mutant that remained at the apical surface.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Transmigration across polarized 16HBE airway epithelial monolayers is a valid model of S. aureus invasion across the human airway epithelium.

  • May combine two claims.

    Findingreasoning

    SpA alone was sufficient to activate RhoA signaling, but by itself did not activate the proteolytic changes in the paracellular junctions unless expressed within the context of intact bacteria.

    “However, although SpA alone was sufficient to activate RhoA signaling, by itself, it did not activate the proteolytic changes in the paracellular junctions unless expressed within the context of intact bacteria.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Transmigration across polarized 16HBE airway epithelial monolayers is a valid model of S. aureus invasion across the human airway epithelium.

  • May combine two claims.

    Findingown data

    SpA+ but not SpA- mutants stimulated activation of EGFR and along with subsequent calpain activity cleaved occludin and E-cadherin, facilitating staphylococcal transmigration through the cell-cell junctions.

    “SpA ؉ but not SpA ؊ mutants stimulated activation of EGFR and along with subsequent calpain activity cleaved the membrane-spanning junctional proteins occludin and E-cadherin, facilitating staphylococcal transmigration through the cell-cell junctions.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Transmigration across polarized 16HBE airway epithelial monolayers is a valid model of S. aureus invasion across the human airway epithelium.

  • May combine two claims.

    Findingown data

    MLC phosphorylation was stimulated in epithelial cells exposed to wild-type S. aureus but not the spa mutant, and SpA alone also induced MLC phosphorylation.

    “Similarly, MLC phosphorylation was stimulated in the epithelial cells exposed to wild-type S. aureus but not the spa mutant (Fig. 3C), and SpA alone also induced MLC phosphorylation (Fig. 3D).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Transmigration across polarized 16HBE airway epithelial monolayers is a valid model of S. aureus invasion across the human airway epithelium.

  • May combine two claims.

    Findingown data

    Wild-type S. aureus strain Newman induces changes in actin distribution and disrupts ZO-1 distribution in polarized human airway epithelial cells.

    “After 24 h of exposure to wild-type S. aureus strain Newman, the distribution of actin was much more heterogeneous, with clumping, regions of discontinuous borders, and overall a much more disorganized pattern of phalloidin staining (Fig. 1A).”
    • Also stated in: § S. aureus Induces Changes in the Distribution of Actin and ZO-1 in Airway Cells- · plain
      “The distribution of the tight junction protein ZO-1 was substantially disrupted in response to the wild-type but not spa mutant staphylococci.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Transmigration across polarized 16HBE airway epithelial monolayers is a valid model of S. aureus invasion across the human airway epithelium.

  • May combine two claims.

    Findingown data

    Blocking EGFR signaling in vivo by pretreating mice with gefitinib did not inhibit the invasive ability of USA300, although EGFR inhibition was documented by decreased induction of ERK1/2 phosphorylation.

    “Blocking EGFR signaling in vivo by pretreating mice with gefitinib (36) did not inhibit the invasive ability of USA300 (Fig. 6E), although we documented EGFR inhibition by demonstrating decreased induction of ERK1/2 phosphorylation (Fig. 6F).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [36] Harari, 2007, J. Clin. Oncol. source

      Not checked in the free run.

    • Rests on an assumption: The mouse model of S. aureus pneumonia is applicable to human invasive pulmonary infection.

  • May combine two claims.

    Interpretationcitation

    EGFR-mediated activation of epithelial proteases, particularly m-calpain through MAPK activity, contributed substantially to bacterial invasion across the tight junctions of the airway epithelium, demonstrated both in vitro and in vivo.

    “EGFR-mediated activation of epithelial proteases, particularly m-calpain through MAPK activity, contributed substantially to bacterial invasion across the tight junctions of the airway epithelium (27), demonstrated both in vitro and in vivo.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [27] Glading, 2004, Mol. Cell. Biol. source

      Not checked in the free run.

    • Rests on an assumption: The mouse model of S. aureus pneumonia is applicable to human invasive pulmonary infection.
    • Rests on an assumption: Transmigration across polarized 16HBE airway epithelial monolayers is a valid model of S. aureus invasion across the human airway epithelium.

  • May combine two claims.

    Interpretationown data

    SpA is necessary but not sufficient to mediate invasive infection across the airway epithelium.

    “Conclusion: SpA is necessary but not sufficient to mediate invasive infection across the airway epithelium.”
    • Also stated in: § SpA Alters the Barrier Properties of Epithelial Monolayers- · hedged
      “These observations indicate that SpA is necessary but not sufficient to induce changes in the epithelial tight junctions.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Transmigration across polarized 16HBE airway epithelial monolayers is a valid model of S. aureus invasion across the human airway epithelium.

  • May combine two claims.

    Interpretationreasoning

    The ability of wild-type staphylococci to invade through the epithelial junction appears to require SpA-mediated induction of both TNF and EGFR signaling and activation of epithelial proteolytic activity.

    “Thus, the ability of wild-type staphylococci to invade through the epithelial junction appears to require SpA-mediated induction of both TNF and EGFR signaling and activation of epithelial proteolytic activity.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Transmigration across polarized 16HBE airway epithelial monolayers is a valid model of S. aureus invasion across the human airway epithelium.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    SpA is a conserved surface protein of all S. aureus strains, highly expressed during the early stages of bacterial growth and abundantly shed from the cell surface.

    “In contrast, protein A (SpA) is a conserved surface protein of all S. aureus strains, highly expressed during the early stages of bacterial growth and abundantly shed from the cell surface (12).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [12] Cheung, 1997, Infect. Immun. source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    SpA binds TNF receptor 1 (TNFR1) and EGF receptor (EGFR).

    “SpA has numerous interactions with host immune effectors binding TNF receptor 1 (TNFR1) (13), EGF receptor (EGFR) (14), IgG (15), and von Willebrand factor (16) as well as activating B cell clonal expansion (17).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [13] Gómez, 2006, J. Biol. Chem. source

      Not checked in the free run.

    • cites [14] Gómez, 2007, EMBO J. source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    SpA has a role in the pathogenesis of murine pneumonia because spa mutants are unable to establish pulmonary infection in a mouse model, and tnfr1-null mice are highly resistant to S. aureus pneumonia.

    “SpA also has a role in the pathogenesis of murine pneumonia because spa mutants are unable to establish pulmonary infection in a mouse model, and tnfr1null mice, lacking the SpA receptor, are highly resistant to S. aureus pneumonia (18).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [18] Gómez, 2004, Nat. Med. source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Staphylococcal toxins are generally expressed during the stationary phase of bacterial growth and may not be present in vivo in sufficient concentration during the early stages of pulmonary infection to participate in staphylococcal invasion.

    “However, staphylococcal toxins are generally expressed during the stationary phase of bacterial growth and may not be present in vivo in sufficient concentration during the early stages of pulmonary infection to participate in staphylococcal invasion (11).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [11] Schmidt, 2001, Infect. Immun. source

      Not checked in the free run.

16 of 16

  • The paper relies on this without support.

    The mechanisms whereby S. aureus invades across the mucosal epithelial barrier to initiate invasive infection are not well understood.

    “The mechanisms whereby these organisms invade across the mucosal epithelial barrier to initiate invasive infection are not well understood.”

  • The paper relies on this without support.

    The mouse model of S. aureus pneumonia is applicable to human invasive pulmonary infection.

    “However, the general lack of susceptibility of mice to S. aureus infection (3) has raised doubts about the applicability of the mouse data to human infection (4).”

  • The paper relies on this without support.

    Transmigration across polarized 16HBE airway epithelial monolayers is a valid model of S. aureus invasion across the human airway epithelium.

    “16HBE cells were grown on 3-m pore size Transwell-Clear filters (Corning-Costar) with an air-liquid interface to form polarized monolayers.”
3 of 3

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Apical inoculum is reported at two different concentrations

    Major

    The transmigration method specifies an apical bacterial concentration of 10^8 cfu/ml, but the results describe the inoculum for that experiment as 1 × 10^9 cfu/ml.

  • ROCK inhibitor pretreatment timing differs

    Minor

    The mouse infection methods specify Y-27632 pretreatment at 24 and 1 hours before inoculation, whereas the Figure 3 legend says it was given 18 hours before infection.

2 of 2

Cites

Flagged first, then by citations

Research works printed but not linked: 3

  1. Hartleib, 2000, Blood, 96, 2149
  2. Finlay, 2005, Curr. Top. Microbiol. Immunol., 291, 1
  3. Martin, 2009, J. Clin. Invest., 119, 1931

Cited by

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Related

Authors
Soong, G., Martin, F. J., Chun, J., Cohen, T. S., Ahn, D. S., & Prince, A.
Affiliations
  • Columbia University Irving Medical Center (US)
  • Columbia University (US)
Corresponding author
Prince, Alice
ORCID
Venue
Journal of Biological Chemistry
Publisher
Elsevier BV
Published
2011-10
Volume
286
Issue
41
Pages
35891-35898
ISSN
0021-9258 (ISSN-L)
DOI
10.1074/jbc.m111.295386
PMC
PMC3195615
OpenAlex
W2046228858
Semantic Scholar
d9fc81c18938f6660f17781123559982b7d3b7e4
Cited by
73
References
43
Citations by year
2026: 2 · 2025: 2 · 2024: 5 · 2023: 1 · 2022: 4 · 2021: 2 · 2020: 6 · 2019: 6 · 2018: 4 · 2017: 8 · 2016: 9 · 2015: 7 · 2014: 3 · 2013: 5 · 2012: 8
FWCI
3.67
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Biological Chemistry (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
Funders
  • National Institutes of Health
  • National Heart, Lung, and Blood Institute (R01HL079395)
Topics
  • Health Sciences › Medicine › Infectious Diseases › Antimicrobial Resistance in Staphylococcus
  • Life Sciences › Immunology and Microbiology › Microbiology › Microbial infections and disease research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
Keywords
Staphylococcus aureus, protein A, bacterial invasion, airway epithelial cells, RhoA/ROCK signaling, calpain, EGF receptor signaling, occludin, E-cadherin, cell-cell contact, methicillin-resistant Staphylococcus aureus, necrotizing pneumonia, MAPK signaling
MeSH terms
Animals, Calpain, Cell Line, Female, Humans, Intercellular Junctions, Mutation, Pneumonia, Staphylococcal, Protease Inhibitors, Staphylococcal Protein A, Staphylococcus aureus, Signal Transduction, Cadherins, Respiratory Mucosa, rho GTP-Binding Proteins, rhoA GTP-Binding Protein, Protein Kinase Inhibitors, Receptors, Tumor Necrosis Factor, Type I, Mice, rho-Associated Kinases, ErbB Receptors

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