Genomic Analyses of Pneumococci from Children with Sickle Cell Disease Expose Host-Specific Bacterial Adaptations and Deficits in Current Interventions

Abstract

Summary Sickle cell disease (SCD) patients are at high risk of contracting pneumococcal infection. To address this risk, they receive pneumococcal vaccines, and antibiotic prophylaxis and treatment. To assess the impact of SCD and these interventions on pneumococcal genetic architecture, we examined the genomes of over 300 pneumococcal isolates from SCD patients over 20 years. Modern SCD strains retained invasive capacity but shifted away from the serotypes used in vaccines. These strains had specific genetic changes related to antibiotic resistance, capsule biosynthesis, metabolism and metal transport. A murine SCD model coupled with Tn-seq mutagenesis identified 60 non-capsular pneumococcal genes under differential selective pressure in SCD, which correlated with aspects of SCD pathophysiology. Further, virulence determinants in the SCD context were distinct from the general population and protective capacity of potential antigens was lost over time in SCD. This highlights the importance of understanding bacterial pathogenesis in the context of high-risk individuals.

The design and analysis of transposon insertion sequencing experiments

“…Bottlenecks are often present in TIS infection studies in which a transposon library is used to infect an animal host 22-26 . For example, it was recently determined that typically only 10 4 -10 5 V. cholerae of 10 9 bacteria inoculated establish infection in a rabbit host, suggesting that >99.99% of bacteria are lost due to infection bottlenecks 27 .…”

“…In general, computational approaches to compensate for bottleneck effects 24-26, 31 determine the fractional loss of insertions in presumed neutral genes during the experiment, then remove a similar fraction of reads from the control library, which enables more accurate comparison between control and experimental datasets. In conditional essentiality analyses, computational compensation for bottlenecks results in substantially fewer false positive assignments and greater confidence in the candidates identified; however, it may also obscure the identification of legitimate conditionally essential insertions (i.e., produce false negatives) if such loci could also be lost solely by chance.…”

“…This scaling, however, assumes that control and experimental datasets have the same complexity, which is not always true, especially in experimental systems with significant bottlenecks. As noted above, several methods have been used to computationally address this problem 24-26, 31 by using the difference in library complexity between TIS datasets to estimate the severity of technical and experimental bottlenecks; the more complex library is then computationally passed through a bottleneck of similar size to model which insertion mutants are likely to be lost entirely by chance during the experiment. Loss resulting from bottlenecks can be simulated by proportionally removing reads equal to the overall difference in diversity between datasets 31 , proportionally removing reads based on the difference in unique reads in a set of known neutral genes 25, 26, 30 or using the difference in overall complexity to simulate reads using a multinomial distribution 24 .…”

Multivalent Pneumococcal Protein Vaccines Comprising Pneumolysoid with Epitopes/Fragments of CbpA and/or PspA Elicit Strong and Broad Protection

“…More recently licensed PCVs have increased serotype coverage (10-and 13-valent) (9,10), but this is at best only a stop-gap measure. The emergence of a high proportion of virulent strains outside the 13-valent vaccine coverage has recently been documented in the high-risk sickle cell disease population (11).…”

Promises and pitfalls of live attenuated pneumococcal vaccines

“…4,6 Even in highly conserved antigens, recombination events can occur whereby strains retain invasive capacity in high-risk hosts or restricted tissue tropisms. 7,8 Such genetic plasticity makes the development of universal pneumococcal vaccines a major scientific challenge, though significant progress has been made in recent years.…”