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Baerga-Ortiz, A.

Publications and source records attributed to Baerga-Ortiz, A..

2 recordsLinked to original sources

The accumulation of colibactin intermediates does not affect the growth and morphology of pks+Escherichia coli

Colibactin is a natural product made by numerous strains of E. coli that harbor the pks genomic island. The deletion of one of the genes within the pks island, the peptidase clbP, has been found to disrupt the maturation of colibactin, thus promoting the accumulation in the periplasmic space of numerous biosynthesis intermediates, some of which have been characterized chemically. To date, no one has reported the effect of such an accumulation of intermediates on the cellular morphology of the producing E. coli bacterium. In this report, we describe the scanning electron microscopy (SEM) images of numerous clinical isolates of E. coli harboring the pks island, collected from Puerto Rico hospitals. We have observed that the wild type isolates that harbor the pks island display lesions on the bacterial envelope surface. These lesions are absent in isolates lacking the pks island. To determine whether this phenotype is associated with colibactin production, we deleted the clbP gene from the extraintestinal pathogenic E. coli strain IHE3034, thus disrupting its ability to make colibactin. The wild-type IHE3034 displayed a spherical shape with no envelope lesions, and was practically indistinguishable from the {Delta}clbP deletion mutant. To our knowledge, this work provides the first SEM images of a pks deletion mutant. ImportanceThe pks genomic island has been linked to the promotion of DNA damage and colorectal cancer, through the production of genotoxic compound colibactin in some strains of E. coli. While much is known about the mechanism of colibactin toxicity once it enters the mammalian cell, the prior steps leading to colibactin secretion or translocation from the bacterial cell, remain unclear. Here, we report high-resolution electron microscopy images of E. coli IHE3034 strain, a known colibactin producer, and a deletion mutant that is known to accumulate colibactin intermediates. The images reveal a predominantly spherical morphology that is unaffected by the accumulation of colibactin precursors and intermediates.

microbiology

A recombinant gp145 Env glycoprotein from HIV-1 expressed in two different cell lines: effects on glycosylation and antigenicity

The envelope glycoprotein (Env) of the human immunodeficiency virus (HIV), has been the primary target for the development of a protective vaccine against infection. The extensive N-linked glycosylation on Env is an important consideration as it may affect efficacy, stability, and expression yields. The expression host has been shown to influence the extent and type of glycosylation that decorates the protein target. Here, we report the glycosylation profile of the candidate subtype C immunogen CO6980v0c22 gp145 produced in two different host cells: CHO-K1 and Expi293F. The amino acid sequence for both glycoproteins was confirmed to be identical by peptide mass fingerprinting. However, the isoelectric point of the proteins differed; 4.5-5.5 and 6.0-7.0 for gp145 produced in CHO-K1 and Expi293F, respectively. These differences in pI were eliminated by enzymatic treatment with sialidase, indicating a large difference in the incorporation of sialic acid between hosts. This dramatic difference in the number of sialylated glycans between hosts was confirmed by analysis of PNGase F-released glycans using MALDI-ToF MS. These differences in glycosylation, however, did not greatly translate into differences in antibody recognition. Biosensor assays showed that gp145 produced in CHO-K1 had similar affinity toward the broadly neutralizing antibodies, 2G12 and PG16, as the gp145 produced in Expi293F. Additionally, both immunogens showed the same reactivity against plasma of HIV-infected patients. Taken together, these results support the notion that there are sizeable differences in the glycosylation of Env depending on the expression host. How these differences translate to vaccine efficacy remains unknown.

biochemistry