Intracellular Bacterial Pathogens Laboratory

Intracellular Bacterial Pathogens Laboratory

Intracellular Bacterial Pathogens Laboratory.

Sónia Castanheira and Francisco García-del Portillo

Intracellular Bacterial Pathogens Laboratory. Penicillium Intracellular Bacterial Pathogens Laboratory. Penicillium (Clutterbuck et al., 1932) and its first purification by Florey, Chain and colleagues (Abraham et al., 1941), confirming its high antibacterial power. Hodgkin would describe its structure a few years later as a molecule containing a beta-lactam ring (Hodgkin, 1949). These researchers received the Nobel Prize for their findings: Fleming, Florey and Chain the Nobel Prize for Medicine in 1945 and Hodgkin for Chemistry in 1964, the latter for the application of X-rays to decipher the structure of important biological molecules.

Once the structure of penicillin was known, interest in knowing its mechanism of action increased. In 1965, Tipper and Strominger proposed a mechanism of action based on the structural analogy of penicillin with the dipeptide D-alanine-D-alanine, present in peptide side chains of peptidoglycan (Tipper and Strominger, 1965). It was also known that penicillin treatment resulted in the accumulation of a UDP-derivative of the disaccharide N-acetyl-muramic (MurNAc)-N-acetyl-glucosamine (Glc-NAc) with a pentapeptide bound to MurNAc. This led to the suspicion that penicillin could inhibit a transpeptidation reaction, involving the terminal part of the lateral pentapeptide (D-Ala-D-Ala).

In the early 1970s, enzymatic activities of the transpeptidase or carboxypeptidase type inhibited by penicillin and, in some cases, morphological effects such as filamentation or loss of bacillary shape were described. In addition, evidence for the stability of the inhibition was accumulating, speculating as to whether the antibiotic was covalently bound (reviewed in Blumberg and Strominger, 1974). However, the identity of these enzymes and the set of enzymes produced by a bacterium were not known. The pioneering work of Spratt and Pardee, published in the journal Nature, made it possible for the first time to simultaneously “visualize” the enzymes that bind beta-lactam antibiotics (Spratt and Pardee, 1975). These researchers incubated a membrane preparation of Escherichia coli with 14C‑benzyl‑penicillin. In the autoradiography several proteins appeared, the "penicillin-binding proteins" (PBPs), which were then assigned numbers from lowest to highest based on decreasing molecular weight. In that same work, they describe the first competition test by previous incubation with a non-radioactive antibiotic, thus being able to determine the relative affinity of any "problem" antibiotic. These simple experiments thus made it possible to associate the binding of the antibiotic to a specific PBP with a morphological effect or a rapid loss of viability. All this represented a great advance in the development of new antibiotics.

Subsequent work by Spratt and colleagues culminated in equally relevant observations: i) all bacteria examined showed several PBPs; and, ii) competition with antibiotics that produced morphological alterations (filamentation, rounding) resulted in E. coli in the disappearance on autoradiography of a single PBP (Spratt, 1975; Spratt, 1977). Thus, the inhibition of PBP3 was associated with the loss of the ability to divide while in the case of PBP2 the ability to elongate peptidoglycan was lost, resulting in the formation of rounded cells (Spratt, 1975; Spratt, 1975). These important functions are, in fact, the basis of the bacteriolytic effect of beta-lactams that show affinity for these PBPs in E. coli in the disappearance on autoradiography of a single PBP (Spratt, 1975; Spratt, 1977). Thus, the inhibition of PBP3 was associated with the loss of the ability to divide while in the case of PBP2 the ability to elongate peptidoglycan was lost, resulting in the formation of rounded cells (Spratt, 1975; Spratt, 1975). These important functions are, in fact, the basis of the bacteriolytic effect of beta-lactams that show affinity for these PBPs in

LINES OF INTEREST AND RESEARCH ACTIVITY

in the disappearance on autoradiography of a single PBP (Spratt, 1975; Spratt, 1977). Thus, the inhibition of PBP3 was associated with the loss of the ability to divide while in the case of PBP2 the ability to elongate peptidoglycan was lost, resulting in the formation of rounded cells (Spratt, 1975; Spratt, 1975). These important functions are, in fact, the basis of the bacteriolytic effect of beta-lactams that show affinity for these PBPs in Salmonella enterica when this pathogen colonizes the interior of the eukaryotic cell to establish a persistent infection. The data we have accumulated show structural alterations of peptidoglycan (PG) that, among other effects, cause a decrease in the signaling capacity mediated by the NF-κB regulator in the host cell (Ramos-Marquès et al., 2017). Therefore, this pathogen appears to "sculpt" the PG to establish a highly successful intracellular lifestyle. An example of this strategy of interference with immune defense systems is a new enzyme used by the intracellular bacterium we call EcgA, which acts as a DL-endopeptidase breaking the bond between D-glutamic and meso-diaminopimelic (D-Glu- mDap) in the peptide side chain of the PG (Rico-Pérez et al., 2016). This D-Glu-mDap configuration (also known as iE-Dap) is key in the recognition of PG fragments by the defense receptor NOD1 (Caruso et al., 2014). Thus, EcgA activity could decrease NOD1 ligand levels in the event that PG fragments are released inside the infected cell.

In addition to EcgA, analysis of the genome of S. enterica In addition to EcgA, analysis of the genome of S. enterica In addition to EcgA, analysis of the genome of Salmonella enterica serovar Typhimurium with deficiencies in PBPs that control cell elongation and division. Note how some of these mutants (upper row) show morphological changes in the intracellular condition (acid pH), while others (lower row) show them in the extracellular condition (neutral pH). historically assumed essential PBP2 and PBP3 (Castanheira et al., 2020). Beta-lactam binding assays indicate that PBP2SAL and PBP3SAL bind antibiotics more effectively at acidic pH while PBP2 and PBP3 do so at neutral pH. Furthermore, PBP3SAL shows low affinity for known beta-lactam antibiotics (Castanheira et al., 2020). This characteristic of PBP3SAL, together with the exchange of PBPs that takes place in the intracellular environment, alerts us to the difficulty of achieving effective therapy for intracellular Salmonella infection using current beta-lactams. In fact, today we know that the exchange of PBP3 for PBP3SAL in intracellular bacteria contributes to the high relapse rate associated with many cases of salmonellosis after termination of antibiotic therapy (Castanheira et al., 2020). This has led us to search for new molecules with high binding affinity to PBP3SAL.

One question underlying these new PBPs is their biological significance. Both PBP2SAL and PBP3SAL apparently perform the same functions in bacterial morphogenesis as those already described for PBP2 and PBP3. So why does the pathogen change them inside the eukaryotic cell, and which regulator(s) does it? These are undoubtedly important questions that we intend to answer. The data we are obtaining allow us to speculate on a possible cross function of these new PBPs with certain important virulence factors in intracellular life. The future will bring us more surprises and questions about these new PBPs, an example of enzymes that are sublimely exploited in an infection cycle by pathogens, but that clearly make their control and eradication difficult for us.

CONTRIBUTION AND SELECTED PUBLICATIONS

1. Abraham EP, Chain E, Fletcher CM, Gardner AD, Heatley NG, Jennings, MA and Florey HW. Abraham EP, Chain E, Fletcher CM, Gardner AD, Heatley NG, Jennings, MA and Florey HW.
2. Abraham EP, Chain E, Fletcher CM, Gardner AD, Heatley NG, Jennings, MA and Florey HW. Abraham EP, Chain E, Fletcher CM, Gardner AD, Heatley NG, Jennings, MA and Florey HW.
3. Abraham EP, Chain E, Fletcher CM, Gardner AD, Heatley NG, Jennings, MA and Florey HW. Abraham EP, Chain E, Fletcher CM, Gardner AD, Heatley NG, Jennings, MA and Florey HW.
4. Abraham EP, Chain E, Fletcher CM, Gardner AD, Heatley NG, Jennings, MA and Florey HW. Abraham EP, Chain E, Fletcher CM, Gardner AD, Heatley NG, Jennings, MA and Florey HW.
5. Castanheira S, Cestero JJ, Rico-Pérez G, García P, Cava F, Ayala JA, Pucciarelli, MG and García-del Portillo, F. Castanheira S, Cestero JJ, Rico-Pérez G, García P, Cava F, Ayala JA, Pucciarelli, MG and García-del Portillo, F.
6. Castanheira S, Cestero JJ, Rico-Pérez G, García P, Cava F, Ayala JA, Pucciarelli, MG and García-del Portillo, F. Castanheira S, Cestero JJ, Rico-Pérez G, García P, Cava F, Ayala JA, Pucciarelli, MG and García-del Portillo, F.
7. Castanheira S, Cestero JJ, Rico-Pérez G, García P, Cava F, Ayala JA, Pucciarelli, MG and García-del Portillo, F. Castanheira S, Cestero JJ, Rico-Pérez G, García P, Cava F, Ayala JA, Pucciarelli, MG and García-del Portillo, F.
8. Fleming A. Fleming A.
9. Fleming A. Fleming A.
10. Fleming A. Fleming A.
11. Fleming A. Fleming A.
12. Fleming A. (2016). A novel peptidoglycan D,L-endopeptidase induced by Salmonella inside eukaryotic cells contributes to virulence. Mol Microbiol 2016;99:546–56.
13. Spratt BG(2016). A novel peptidoglycan D,L-endopeptidase induced by Salmonella inside eukaryotic cells contributes to virulence. Mol Microbiol 2016;99:546–56.
14. (2016). A novel peptidoglycan D,L-endopeptidase induced by Salmonella inside eukaryotic cells contributes to virulence. Mol Microbiol 2016;99:546–56. (2016). A novel peptidoglycan D,L-endopeptidase induced by Salmonella inside eukaryotic cells contributes to virulence. Mol Microbiol 2016;99:546–56.
15. (2016). A novel peptidoglycan D,L-endopeptidase induced by Salmonella inside eukaryotic cells contributes to virulence. Mol Microbiol 2016;99:546–56. (2016). A novel peptidoglycan D,L-endopeptidase induced by Salmonella inside eukaryotic cells contributes to virulence. Mol Microbiol 2016;99:546–56.
16. (2016). A novel peptidoglycan D,L-endopeptidase induced by Salmonella inside eukaryotic cells contributes to virulence. Mol Microbiol 2016;99:546–56. (2016). A novel peptidoglycan D,L-endopeptidase induced by Salmonella inside eukaryotic cells contributes to virulence. Mol Microbiol 2016;99:546–56.