Identification of a cytotoxic factor from a non-pigmented entomopathogenic Serratia marcescens isolate toxic towards human carcinoma cell lines.

It has been reported that cell-free culture broths and some proteins from pigmented and non-pigmented Serratia spp. are cytotoxic towards cancerous and non-cancerous human cell lines. Looking for new molecules toxic against human cancerous cells but harmless towards normal human cells, the aim of this work was (a) to determine whether cell-free broths from the entomopathogenic non-pigmented S. marcescens 81 (Sm81), S. marcescens 89 (Sm89) and S. entomophila (SeMor4.1) presented cytotoxic activity towards human carcinoma cell lines; (b) to identify and purify the associated cytotoxic factor(s) and (c) to evaluate whether the cytotoxic factor(s) was cytotoxic towards non-cancerous human cells. This research was focussed on the observed morphology changes and the proportion of remaining viable cells after incubation in the presence of cell-free culture broths from the Serratia spp isolates to evaluate cytotoxic activity. The results showed that broths from both S. marcescens isolates presented cytotoxic activity and induced cytopathic-like effects on the human neuroblastoma CHP-212 and the breast cancer MDA-MB-231 cells. Slight cytotoxicity was observed in the SeMor4.1 broth. A serralysin-like protein of 50 kDa was identified in Sm81 broth as responsible for cytotoxic activity after purification by ammonium sulphate precipitation and ion-exchange chromatography followed by tandem-mass spectrometry (LC-MS/MS). The serralysin-like protein was toxic against CHP-212 (neuroblastoma), SiHa (human cervical carcinoma) and D-54 (human glioblastoma) cell lines in a dose-dependent manner and showed no cytotoxic activity in primary cultures of normal non-cancerous human keratinocytes and fibroblasts. Therefore, this protein should be evaluated for a potential use as an anticancer agent.

Pathogenicity of Isolates of Serratia Marcescens towards Larvae of the Scarab Phyllophaga Blanchardi (Coleoptera).

Serratia marcescens is a Gram negative bacterium (Enterobacteriaceae) often associated with infection of insects. In order to find pathogenic bacteria with the potential to control scarab larvae, several bacterial strains were isolated from the hemocoel of diseased Phyllophaga spp (Coleoptera:Scarabaeidae) larvae collected from cornfields in Mexico. Five isolates were identified as Serratia marcescens by 16S rRNA gene sequencing and biochemical tests. Oral and injection bioassays using healthy Phyllophaga blanchardi larvae fed with the S. marcescens isolates showed different degrees of antifeeding effect and mortality. No insecticidal activity was observed for Spodoptera frugiperda larvae (Lepidoptera: Noctuidae) by oral inoculation. S. marcescens (Sm81) cell-free culture supernatant caused significant antifeeding effect and mortality to P. blanchardi larvae by oral bioassay and also mortality by injection bioassay. Heat treated culture broths lost the ability to cause disease symptoms, suggesting the involvement of proteins in the toxic activity. A protein of 50.2 kDa was purified from the cell-free broth and showed insecticidal activity by injection bioassay towards P. blanchardi. Analysis of the insecticidal protein by tandem- mass spectrometry (LC-MS/MS) showed similarity to a Serralysin-like protein from S. marcescens spp. This insecticidal protein could have applications in agricultural biotechnology.

Identification of a putative Mexican strain of Serratia entomophila pathogenic against root-damaging larvae of Scarabaeidae (Coleoptera).

The larvae of scarab beetles, known as "white grubs" and belonging to the genera Phyllophaga and Anomala (Coleoptera: Scarabaeidae), are regarded as soil-dwelling pests in Mexico. During a survey conducted to find pathogenic bacteria with the potential to control scarab larvae, a native Serratia sp. (strain Mor4.1) was isolated from a dead third-instar Phyllophaga blanchardi larva collected from a cornfield in Tres Marías, Morelos, Mexico. Oral bioassays using healthy P. blanchardi larvae fed with the Mor4.1 isolate showed that this strain was able to cause an antifeeding effect and a significant loss of weight. Mortality was observed for P. blanchardi, P. trichodes, and P. obsoleta in a multidose experiment. The Mor4.1 isolate also caused 100% mortality 24 h after intracoelomic inoculation of the larvae of P. blanchardi, P. ravida, Anomala donovani and the lepidopteran insect Manduca sexta. Oral and injection bioassays were performed with concentrated culture broths of the Mor4.1 isolate to search for disease symptoms and mortality caused by extracellular proteins. The results have shown that Mor4.1 broths produce significant antifeeding effects and mortality. Mor4.1 broths treated with proteinase K lost the ability to cause disease symptoms and mortality, in both the oral and the injection bioassays, suggesting the involvement of toxic proteins in the disease. The Mor4.1 isolate was identified as a putative Serratia entomophila Mor4.1 strain based on numerical taxonomy and phylogenetic analyses done with the 16S rRNA gene sequence. The potential of S. entomophila Mor4.1 and its toxins to be used in an integrated pest management program is discussed.