Bacteria exhibit a myriad of different morphologies, through the synthesis and modification of their essential peptidoglycan (PG) cell wall. Our discovery of a fluorescent D-amino acid (FDAA)-based PG labeling approach provided a powerful method for observing how these morphological changes occur. Given that PG is unique to bacterial cells and a common target for antibiotics, understanding the precise mechanism(s) for incorporation of (F)DAA-based probes is a crucial determinant in understanding the role of PG synthesis in bacterial cell biology and could provide a valuable tool in the development of new antimicrobials to treat drug-resistant antibacterial infections. Here, we systematically investigate the mechanisms of FDAA probe incorporation into PG using two model organisms Escherichia coli (Gram-negative) and Bacillus subtilis (Gram-positive). Our in vitro and in vivo data unequivocally demonstrate that these bacteria incorporate FDAAs using two extracytoplasmic pathways: through activity of their D,D-transpeptidases, and, if present, by their L,D-transpeptidases and not via cytoplasmic incorporation into a D-Ala-D-Ala dipeptide precursor. Our data also revealed the unprecedented finding that the DAA-drug, D-cycloserine, can be incorporated into peptide stems by each of these transpeptidases, in addition to its known inhibitory activity against D-alanine racemase and D-Ala-D-Ala ligase. These mechanistic findings enabled development of a new, FDAA-based, in vitro labeling approach that reports on subcellular distribution of muropeptides, an especially important attribute to enable the study of bacteria with poorly defined growth modes. An improved understanding of the incorporation mechanisms utilized by DAA-based probes is essential when interpreting results from high resolution experiments and highlights the antimicrobial potential of synthetic DAAs.

Objective: To investigate the applicability of PLCζ analysis in assisting the clinical decisionmaking process when considering artificial oocyte activation (AOA) for infertile males in assisted reproductive technology (ART). Design:To screen 46 males (43 infertile/13 fertile) using our PLCζ assay.Setting: Fertility unit/university laboratory.Patients: Infertile males with (1) abnormal sperm morphology, (2) total fertilization failure, low fertilization rate (<50%), or repeated fertilization failure in ART. Intervention(s):We analysed PLCζ levels in sperm from fertile and infertile males. Eligible patients subsequently underwent ICSI/AOA with calcimycin (GM508). Main Outcome Measure(s): PLCζ localization, level and the proportion of sperm expressingPLCζ. Thresholds of PLCζ-deficiency, fertilization rates, pregnancy rates and live birth rates of AOA and non-AOA cycles.Results: Compared with 13 controls, 34 of the 43 infertile males had significantly lower levels of PLCζ and/or a significantly lower proportion of sperm exhibiting PLCζ. Of these 34 patients, 15 showed a significant PLCζ reduction in both parameters, which we termed 'PLCζ deficiency'. Five PLCζ-deficient patients opted for AOA; all 5 achieved fertilization and 4 achieved clinical pregnancies and live births. Fertilization rate improved significantly from 18.6% (intracytoplasmic sperm injection, ICSI) to 56.8% (ICSI/AOA) (p<0.001). The clinical pregnancy rate and live birth rate with AOA were both 40% per initiated cycle. Youden index analysis revealed that the cut-offs below which infertile males were likely to benefit from AOA were 71% for the proportion of sperm expressing PLCζ and 15.57 arbitrary units for mean PLCζ level. Conclusion:PLCζ analysis is a useful diagnostic tool to determine patient eligibility for subsequent AOA treatment.

Oocyte activation deficiency (OAD) remains the predominant cause of total/low fertilization rate in assisted reproductive technology (ART). Phospholipase C zeta (PLCζ) is the dominant sperm-specific factor responsible for triggering oocyte activation in mammals. OAD has been linked to numerous PLCζ abnormalities in patients experiencing failed in-vitro fertilization (IVF) or intra-cytoplasmic sperm injection (ICSI) cycles. While significant efforts have enhanced our understanding of the clinical relevance of PLCζ, and the potential effects of genetic variants upon functionality, our ability to apply PLCζ in a diagnostic or therapeutic role remains limited. Artificial oocyte activation (AOA) is the only option for patients experiencing OAD but lacks a reliable diagnostic approach. Immunofluorescence analysis has revealed that the levels and localization patterns of PLCζ within sperm can help us to indirectly diagnose a patient’s ability to induce oocyte activation. Screening of the gene encoding PLCζ protein is also critical if we are to fully determine the extent by which genetic factors might play a role in the aberrant expression and/or localization patterns observed in infertile patients. Collectively, these findings highlight the clinical potential of PLCζ, both as a prognostic indicator of OAD, and eventually as a therapeutic agent. In this review, we focus on our understanding of the association between OAD and PLCζ by discussing the localization and expression of this key protein in human sperm, the potential genetic causes of OAD, and the diagnostic tools that are currently available to us to identify PLCζ deficiency and select patients that would benefit from targeted therapy.

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