Glutamine Synthetase and Glucose-6-Phosphate Isomerase Are Adhesive Moonlighting Proteins of Lactobacillus crispatus Released by Epithelial Cathelicidin LL-37

Abstract

c Glutamine synthetase (GS) and glucose-6-phosphate isomerase (GPI) were identified as novel adhesive moonlighting proteins of Lactobacillus crispatus ST1. Both proteins were bound onto the bacterial surface at acidic pHs, whereas a suspension of the cells to pH 8 caused their release into the buffer, a pattern previously observed with surface-bound enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of L. crispatus. The pH shift was associated with a rapid and transient increase in cell wall permeability, as measured by cell staining with propidium iodide. A gradual increase in the release of the four moonlighting proteins was also observed after the treatment of L. crispatus ST1 cells with increasing concentrations of the antimicrobial cationic peptide LL-37, which kills bacteria by disturbing membrane integrity and was here observed to increase the cell wall permeability of L. crispatus ST1. At pH 4, the fusion proteins His 6 -GS, His 6 -GPI, His 6 -enolase, and His 6 -GAPDH showed localized binding to cell division septa and poles of L. crispatus ST1 cells, whereas no binding to Lactobacillus rhamnosus GG was detected. Strain ST1 showed a pH-dependent adherence to the basement membrane preparation Matrigel. Purified His 6 -GS and His 6 -GPI proteins bound to type I collagen, and His 6 -GS also bound to laminin, and their level of binding was higher at pH 5.5 than at pH 6.5. His 6 -GS also expressed a plasminogen receptor function. The results show the strain-dependent surface association of moonlighting proteins in lactobacilli and that these proteins are released from the L. crispatus surface after cell trauma, under conditions of alkaline stress, or in the presence of the antimicrobial peptide LL-37 produced by human cells.

Network of Surface-Displayed Glycolytic Enzymes in Mycoplasma pneumoniae and Their Interactions with Human Plasminogen

“…In addition to PdhA, PdhB, and GapA, other glycolytic enzymes such as lactate dehydrogenase (Ldh) are detected in the Triton X-100-insoluble fraction that represents the membrane-associated proteins of M. pneumoniae (28). Finally, additional glycolytic enzymes are described in other bacterial species but have not hitherto been characterized in mycoplasmas as surface displayed, indicating a role for other proteins as potential interaction partners with host factors (29)(30)(31)(32). In summary, the present information regarding surface-localized glycolytic enzymes suggests a complex network of interactions with different host factors, which may be important in understanding the pathogenesis of M. pneumoniae infections.…”

BopA Does Not Have a Major Role in the Adhesion of Bifidobacterium bifidum to Intestinal Epithelial Cells, Extracellular Matrix Proteins, and Mucus

“…The immunofluorescence staining was used to confirm the presence of BopA on the surface of B. bifidum as detailed previously (21). Briefly, B. bifidum cells were cultivated, washed with phosphate-buffered saline (PBS), and fixed with 3.5% (wt/vol) paraformaldehyde in PBS prior labeling with anti-BopA antiserum or preimmune serum as the primary antiserum and Alexa-488 (Invitrogen)-conjugated anti-rabbit IgG (1 g ml Ϫ1 ) as the secondary antibody.…”

Multitasking Bacterial Moonlighting Proteins in Bacterial Virulence and Infectious Disease

“…However, Boel et al [21] suggest that there is strong evidence that the cell surface enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of S.pyogenes is there as part of an undescribed system for secreting cytoplasmic enzymes. This hypothesis is supported by the studies of the surface location of GAPDH in Lactobacillus crispatus, which reveals that soluble GAPDH does not bind to the surface of the intact organism [22].…”