Impact of Escherichia coli K12 and O18:K1 on human platelets: Differential effects on platelet activation, RNAs and proteins.

Blood platelets can interact with bacteria, possibly leading to platelet activation, cytokine and microparticle release and immune signalling. Besides, bacteria can also affect the platelet RNA content. We investigated the impact of non-pathogenic K12 and pathogenic O18:K1 Escherichia (E.) coli strains on platelet activation, RNA expression patterns, and selected proteins. Depending on bacteria concentration, contact of platelets with E. coli K12 lead to an increase of P-selectin (24-51.3%), CD63 (15.9-24.3%), PAC-1 (3.8-14.9%) and bound fibrinogen (22.4-39%) on the surface. E. coli O18:K1 did not affect these markers. Sequencing analysis of total RNA showed that E. coli K12 caused a significant concentration change of 103 spliced mRNAs, of which 74 decreased. For the RNAs of HMBS (logFC = +5.73), ATP2C1 (logFC = -3.13) and LRCH4 (logFC = -4.07) changes were detectable by thromboSeq and Tuxedo pipelines. By Western blot we observed the conversion of HMBS protein from a 47 kDA to 40 kDa product by E. coli K12, O18:K1 and by purified lipopolysaccharide. While ATP2C1 protein was released from platelets, E. coli either reduced the secretion or broke down the released protein making it undetectable by antibodies. Our results demonstrate that different E. coli strains influence activation, RNA and protein levels differently which may affect platelet-bacteria crosstalk.

Platelet RNA as a circulating biomarker trove for cancer diagnostics.

Platelets are multifunctional cell fragments, circulating in blood in high abundance. Platelets assist in thrombus formation, sensing of pathogens entering the blood stream, signaling to immune cells, releasing vascular remodeling factors, and, negatively, enhancing cancer metastasis. Platelets are 'educated' by their environment, including in patients with cancer. Cancer cells appear to initiate intraplatelet signaling, resulting in splicing of platelet pre-mRNAs, and enhance secretion of cytokines. Platelets can induce leukocyte and endothelial cell modeling factors, for example, through adenine nucleotides (ATP), thereby facilitating extravasation of cancer cells. Besides releasing factors, platelets can also sequester RNAs and proteins released by cancer cells. Thus, platelets actively respond to queues from local and systemic conditions, thereby altering their transcriptome and molecular content. Platelets contain a rich repertoire of RNA species, including mRNAs, small non-coding RNAs and circular RNAs; although studies regarding the functionality of the various platelet RNA species require more attention. Recent advances in high-throughput characterization of platelet mRNAs revealed 10 to > 1000 altered mRNAs in platelets in the presence of disease. Hence, platelet RNA appears to be dynamically affected by pathological conditions, thus possibly providing opportunities to use platelet RNA as diagnostic, prognostic, predictive, or monitoring biomarkers. In this review, we cover the literature regarding the platelet RNA families, processing of platelet RNAs, and the potential application of platelet RNA as disease biomarkers.

Isolation of a herpes simplex virus type 1 mutant deleted for the essential UL42 gene and characterization of its null phenotype.

We isolated a cell line, designated V9, stably transformed with the herpes simplex virus type 1 (HSV-1) UL42 gene, which is one of seven genes required in trans for the replication of plasmids containing an HSV origin of replication (C. A. Wu, N. J. Nelson, D. J. McGeoch, and M. D. Challberg, J. Virol. 62:435-443, 1988). V9 cells inducibly expressed the product of the UL42 gene, the 65-kDa DNA-binding protein (65KDBP), and were used as a permissive host to construct a mutant virus deleted for this essential gene. The UL42 deletion mutant, designated Cgal delta 42, displayed a tight early phenotype in nonpermissive Vero cells producing no infectious progeny, viral DNA, or late gene products but accumulated selected immediate-early and early transcripts with kinetics similar to those of wild-type virus. Wild-type levels of viral DNA and infectious progeny were produced in permissive V9 cells, despite the fact that V9 cells infected with Cgal delta 42 accumulated less than 1% of the UL42 RNA and protein found in Cgal+ virus-infected V9 or Vero cells. These results indicate that only small quantities of the 65KDBP are required for the synthesis of HSV DNA and the production of infectious virus. Although we could find no evidence that the superinduction of the 65KDBP in V9 cells infected with Cgal+ repressed expression of HSV-1 genes as observed in cells expressing another DNA-binding protein, ICP8 (P. K. Orberg and P. A. Schaffer, J. Virol. 61:1136-1146, 1987), the induction of the 65KDBP in V9 cells correlated with an approximately 2-h-earlier shift in the expression of genes from all three kinetic classes. The availability of the UL42 mutant should facilitate the construction of more subtle UL42 mutants which will be useful in the elucidation of the interrelationship between the 65KDBP and other DNA replication proteins as well as in the characterization of additional important functional domains.

Susceptibility of members of the family Legionellaceae to thermal stress: implications for heat eradication methods in water distribution systems.

To ascertain the feasibility of heat inactivation as an eradication method applicable to all members of the family Legionellaceae, we tested the heat resistance of 75 isolates which represented 19 members of this family of organisms. The ranges of thermal death times at 60, 70, and 80 degrees C were 1.3 to 10.6, 0.7 to 2.6, and 0.3 to 0.7 min, respectively. These data suggest that the method of heat eradication will be effective against all members of the family Legionellaceae.

A note on symbiosis of Legionella pneumophila and Tatlockia micdadei with human respiratory flora.

Sixteen micro-organisms, representing clinically important respiratory microflora, were tested for their ability to stimulate the growth of Legionella pneumophila and Tatlockia micdadei in nutritionally-deficient agar media. Nutritional symbiosis, indicated by the appearance of satellite colonies of L. pneumophila or T. micdadei, was observed for H. influenzae and N. meningitidis. This interaction between normal flora and pathogens of the respiratory tract may have clinical relevance in the pathogenesis of Legionnaires' disease and Pittsburgh pneumonia.

Nosocomial Legionella pneumonia in a population of head and neck cancer patients.

A prospective study of nosocomial pneumonia following major head and neck surgery was conducted when it was recognized that Legionella contaminated the hospital water supply. Legionella pneumonia had not previously been diagnosed in our hospital. Every head and neck patient with nosocomial pneumonia had specialized tests performed. During the 18-month study period, 29% of patients with nosocomial pneumonia (7 of 27) had evidence of legionellosis. The sero-group of the infecting Legionella was the same as the Legionella in the water supply. Legionella was seen exclusively in patients with clinically evident aspiration. Legionella pneumonia was not demonstrated in patients undergoing laryngectomy. We conclude that specialized testing must be employed to avoid delay in diagnosis and failure to administer specific antibiotic therapy. Legionellosis may be underdiagnosed in hospitalized patients.

Nosocomial legionellosis in surgical patients with head-and-neck cancer: implications for epidemiological reservoir and mode of transmission.

A prospective pneumonia study was conducted simultaneously on head-and-neck surgery wards at two hospitals over 2 years; one hospital had a water supply contaminated with Legionella pneumophila but no record of having had a case of legionella pneumonia, and the other had just decontaminated its water supply because of known endemic nosocomial legionellosis. Special laboratory tests for legionella were done on all cases of nosocomial pneumonia irrespective of clinical impression. Over the first 18 months, the rate of nosocomial legionellosis was 30% at the first hospital and 0% at the second. Patients who underwent laryngectomy did not acquire the disease. Hyperchlorination at the first hospital was followed by a fall (p less than 0.01) in legionella pneumonias. Thus legionella pneumonias can be overlooked if special laboratory tests are not applied routinely, and surgical patients with head-and-neck cancer may be at high risk of nosocomial legionellosis because of the potential for pulmonary aspiration of contaminated water or orophyaryngeal microflora and/or frequent manipulation of the respiratory tract. This study demonstrates the benefits of examining the environment for legionella despite the absence of documented disease.

Tatlockia micdadei (Pittsburgh pneumonia agent) growth kinetics may explain its infrequent isolation from water and the low prevalence of Pittsburgh pneumonia.

Sediment and indigenous microflora taken from water distribution systems has been shown to promote the survival of Legionella pneumophila. The effect of sediment and indigenous microflora on Tatlockia micdadei (Pittsburgh pneumonia agent, PPA) was evaluated by growth curve experiments. Symbiosis between PPA and environmental bacteria was demonstrated by satellitism experiments. Unlike L. pneumophila, the concentration of PPA remained stationary in test tube suspensions containing both microflora and sediment. The difference in the ecology between the two organisms may explain the infrequent environmental recovery of PPA and, ultimately, the epidemiologic differences between Legionnaires disease and Pittsburgh pneumonia.

Ecology of Legionella pneumophila within water distribution systems.

The reservoir for hospital-acquired Legionnaires disease has been shown to be the potable water distribution system. We investigated the influence of the natural microbial population and sediment (scale and organic particulates) found in water systems as growth-promoting factors for Legionella pneumophila. Our in vitro experiments showed that: (i) water from hot-water storage tank readily supported the survival of L. pneumophila, (ii) the concentration of sediment was directly related to the survival of L. pneumophila, (iii) the presence of environmental bacteria improved the survival of L. pneumophila via nutritional symbiosis, (iv) the combination of sediment and environmental bacteria acted synergistically to improve the survival of L. pneumophila, and (v) the role of sediment in this synergistic effect was determined to be nutritional. Sediment was found to stimulate the growth of environmental microflora, which in turn stimulated the growth of L. pneumophila. These findings confirm the empiric observations of the predilection of L. pneumophila for growth in hot-water tanks and its localization to sediment. L. pneumophila occupies an ecological niche within the potable water system, with interrelationships between microflora, sediment, and temperature.