Detection of Penicillin‐binding Proteins in the Endosymbiont of the Trypanosomatid Crithidia deanei

Abstract

ABSTRACT. Growth by serial transfers of the trypanosomatid Crithidia deanei in culture medium containing 1 mg/ml of the β‐lactam antibiotics ampicillin or cephalexin resulted in shape distortion of its endosymbiont. The endosymbiont first appeared as filamentous structures with restricted areas of membrane damage. An increase of electron lucid areas was also observed in the endosymbiont matrix. The continuous treatment with β‐lactam antibiotics, resulted in endosymbiont membranes fragmentation; and later on the space previously occupied by the symbiont was identified as an electron lucid area in the host cytoplasm. The putative targets of β‐lactam antibiotic were two membrane‐bound penicillin‐binding proteins (PBPs) detected in the Sarkosyl‐soluble fraction of purified symbionts labeled with [3H]‐benzylpenicillin. The apparent molecular weight of the proteins were 90 kDa (PBP1) and 45 kDa (PBP2). PBP2 represented 85% of the total PBP content in the membrane fraction of the endosymbionts. Competition experiments using the tested antibiotics and [3H]‐benzylpenicillin showed that ampicillin and cephalexin have half saturating concentrations considerably higher than [3H]‐benzylpenicillin and indicated that PBP1 is the probable lethal target of the antibiotics tested. These results suggest that a physiologically active PBP is present in the cell envelope of C. deanei endosymbionts and may play important roles in the control of processes such as cell division and shape determination.

Predicting the Proteins of Angomonas deanei, Strigomonas culicis and Their Respective Endosymbionts Reveals New Aspects of the Trypanosomatidae Family

“…The peptidoglycan layer is functional in trypanosomatid symbionts, as shown by treatment with β-lactam antibiotics affecting the division of the bacterium, generating filamentous structures and culminating in cell lysis. PBP1 and PBP2 have also been detected at the symbiont envelope [6]. PBP1B interacts with the two essential division proteins, FtsN and PBP3/FtsI, which are absent in the symbiont.…”

“…Moreover, the symbiont envelope contains a reduced peptidoglycan layer and lacks a septum during its division process, which can be related to the facilitation of metabolic exchanges, as well as to the control of division by the host protozoan [6]. These losses could be understood since the host trypanosomatid is controlling the number of symbiotic bacteria per cell.…”

“…In this obligatory association, the endosymbiont is unable to survive and replicate once isolated from the host, whereas aposymbiotic protozoa are unable to colonize insects [4], [5]. The symbiont is surrounded by two membrane units and presents a reduced peptidoglycan layer, which is essential for cell division and morphological maintenance [6]. The lack of a typical gram-negative cell wall could facilitate the intense metabolic exchange between the host cell and the symbiotic bacterium.…”

Endosymbiosis in protozoa of the Trypanosomatidae family

“…The same approach has been used by other authors, so that at present, cultures of wild and aposymbiotic strains of B. culicis, C. deanei, C. oncopelti, and Herpetomonas roitmani are available. Recently Motta et al [13] carried out a detailed study on the e¡ect the of L-lactam antibiotics, ampicillin and cephalexin, on the C. deanei endosymbiont. Electron microscopic analysis showed that the endosymbionts su¡ered a progressive shape distortion process, leading to the appearance of pleomorphic forms.…”

“…Recent studies using isolated endosymbionts from C. deanei showed the presence of two penicillin-binding proteins (PBP) in the envelope of these microorganisms with apparent molecular weights of 90 and 45 kDa [13]. The treatment with L-lactam antibiotics, leads to morphological alterations in the endosymbionts, indicating that a reduced or degenerated cell wall may exist and play important physiological roles in the division process and in morphological integrity maintenance [13]. The endosymbiont matrix has two distinct areas, one electron dense and the other electron lucid.…”