University of Groningen
Quantifying the transcriptome of a human pathogen
Aprianto, Rieza
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2018
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Aprianto, R. (2018). Quantifying the transcriptome of a human pathogen: Exploring transcriptional
adaptation of Streptococcus pneumoniae under infection-relevant conditions. Rijksuniversiteit Groningen.
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1
The ancien
t sc
our
ge
Streptococcus pneumoniae: the ancient scourge of
modern society
Five centuries before the Common Era, Hippocrates of Kos (c. 460–367 B.C.), the Father of Medicine, described the diagnosis and remedy for
pneu-monia — a form of infection into the lower respiratory tract1. In his
in-fluential corpus, he referred to pneumonia as “those which the ancients named”, exemplifying that the scourge of pneumonia was known even by earlier societies than the Ancient Greeks. Two and a half millennia later, lower respiratory tract infections (LRTIs) are still very much a part of our modern society. A recent report showed that LRTIs are the deadliest
communicable disease and the fifth most common cause of global death2.
In addition, the infections cause principal loss of healthy life (disability- adjusted life years, i.e.: a combination of mortality and morbidity), right
behind ischemic heart disease3. Pneumonia, an infection of the lung
alve-oli4 is the most important form of lower respiratory tract infection.
The most prominent etiologic agent of pneumonia is the Gram posi-tive opportunistic pathogen Streptococcus pneumoniae. This bacterium is responsible for the majority of LRTIs cases while single-handedly
plac-ing LRTIs as the deadliest infectious disease2. Aside from pneumonia,
S. pneumoniae causes milder infections, such as otitis media and sinusitis,
and other severe and lethal infections, including meningitis and
septice-mia5. These pneumococcal infections are distinguished by high mortality
rates in young children with 59% of pneumococcal meningitis cases and 45% of septicemia cases resulting in death. In particular, pneumococcal- related mortality is higher in African children than in children from other
continents6. Although developing countries tend to bear the
pneumococ-cal brunt, developed wealthy societies recently reported high incidence of
pneumococcal infections in the elderly population7, making the
pneumo-coccus a general health issue to all human populations.
In most cases, pneumococcus resides in the host nasopharyngeal passage without symptoms. In fact, S. pneumoniae is part of the
typ-ical microbiota of the upper respiratory tract8–10. Pneumococcal
car-riage begins in the first two years of life11 and colonization rates depend
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people in a household13, day-care attendance14, number of other
chil-dren15, bed-sharing and malnutrition16. This asymptomatic colonization
is a prerequisite for further pneumococcal infections17. Because of its
im-pact on general health, vaccination programs against the pneumococcus have been introduced. Unfortunately, limited success has been reported on these programs, with vaccine-target strains being replaced by non-
vaccine strains capable of causing invasive infections5.
In the 1940s, the treatment of pneumococcal pneumonia greatly
bene-fitted from the introduction of sulfonamides and penicillin in the clinics18.
However, pneumococcal resistance to penicillin and other antimicrobials
quickly spread worldwide19. Soon afterwards, resistance to more than one
antibiotic was reported in S. pneumoniae and, more worryingly, half of invasive pneumococcal cases in the United States were resistant to at least
one antibiotic20. In addition, pneumococcal resistance to a wide range of
clinically-relevant antibiotics has been reported around the globe21,22.
Unlikely help: pneumococcus assisting biological
research
Five years after Sternberg23 and Pasteur24 reported independently the
pathogenic potential of S. pneumoniae, Fraenekel25 called the bacterium
the pneumococcus due to its propensity for causing pneumonia. Later, the
bacterium was renamed Diplococcus pneumoniae by the Society of
Amer-ican Microbiologists26, referring to its characteristic shape under the
mi-croscope which resembles a pair of cocci. Finally, in 1974, the
pneumococ-cus was reclassified under the genus Streptococcpneumococ-cus27. Since its discovery,
the bacterium has been the subject of seminal breakthroughs, including
its role in the discovery of Gram staining28, in the demonstration of
anti-genic properties of polysaccharides29, and the first successful case of
pen-icillin treatment in clinical infection30.
The most influential role of the pneumococcus in biological research is the conclusive evidence that DNA exclusively carries the genetic code (later RNA-based virus was discovered to be the exception to this rule). Griffith was the first to show that phenotype, in this specific case,
1
Comple x biologic al s ys temexpression of a capsule can be transferred from capsule-producing strain
to non-capsular S. pneumoniae inside a murine host31. The result was then
verified, expanded and optimized32–35. Avery et al. built on these
observa-tions and fine-tuned the method to determine that DNA is the material which mediated the phenotypic transfer between the two pneumococcal
strains36. We now recognize that all known capsule genes except in one
pneumococcal serotype are encoded in a single operon, the cps operon,
which is located between two conserved genes: dexB and aliA37. The
ge-nomic arrangement of the capsule mediates easy and efficient transfer be-tween strains and, in turn, facilitates capsule switching among different serotypes. We now understand that by exploiting this strategy, S.
pneumo-niae may escape vaccine-induced immune pressure38.
Biological systems, genome-wide approach and
RNA-sequencing
Reductionism has been a successful and crucial approach in molecular bi-ology, due to its ability to connect a gene or set of genes (genotype) to a measurable trait (phenotype). Reductionists clarified that the presence of a pneumococcal capsule and its serotype are determined by the presence and precise genotype of the cps operon. Unfortunately, reductionism fails to explain complicated biological phenomena, including interspecies inter-action, immunity and infection. In the last decade, reductionism has been conceding to holism, a perspective that considers interactions between ev-ery component of the system. This approach appreciates the continuous interaction between every component, biotic and abiotic, and their simul-taneous modification. Furthermore, while the environment constraints bi-ological components, the living component changes the environment to
its needs39, emphasizing the transient and dynamic nature of the system.
The complexity of this system causes novel properties to emerge, which in
turn, determines the direction and characteristics of the system40.
Bacterial infection is a classic example of such complex biological sys-tem. In infection sites, the pathogen multiplies and acquires nutrients to fuel its expansion while simultaneously evading the immune response.
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On the other hand, the host struggles to remove invading pathogens by innate immune responses and specialized cells. The pathogen and the host interact intimately and alter the environment according to their re-spective needs. In order to elucidate emergent properties of such complex system and to have a bird eye’s view over the phenomenon, we need to
exhaustively measure every parameters in a comprehensive manner41.
In addition, the shift of the paradigm has been spurred by cutting edge advances, especially in sequencing technology and processing large bio-logical datasets. Transcriptomics, for example, allows researchers unprece-dented access to the genome-wide transcriptome and to the way it changes during a specific phenomenon. In the last four decades, sequencing tech-nology has been perfected into its current high- throughput incarnation. When introduced, sequencing was used to decipher the genetic code (ge-nomics) and then when coupled with cDNA generation, it was employed to
decipher the transcriptome (transcriptomics), through RNA-seq42. Because
of improvements in library preparation and sequencing efficiency, more nucleic bases can be sequenced in less time, driving down cost and
justi-fying sequencing as a routine protocol43. Prior to the widespread use of
se-quencing-based technology, array-based technologies, such as microarray and tiling array, were the platform of choice for transcriptomics studies. Compared to array-based approach, RNA-seq has a wider dynamic range, resulting in better detection of transcript boundaries and more powerful
differential expression analysis44.
Furthermore, recent reports dispel the myth of the simplicity of the prokaryotic transcriptome. Rather, the bacterial transcriptome is as
com-plex as eukaryotic transcriptome45,46. In addition, sequencing results have
permitted the elucidation of genomics architecture and regulatory struc-tures of gene expression. For example, bacteria employ a wide range of
small RNAs to regulate gene expression, both cis- and trans-acting47.
Ad-ditionally, multiple start sites permit alternative forms of operon48,
fur-ther expanding its genomic potential. As an illustration, the genome-wide examination of the small bacterium Helicobacter pylori (1.7 Mbp) showed ubiquitous transcription start sites: inside and opposite of coding
sequences and numerous sRNAs49 generating an ample genomic
1
Subtle virulenc
e
features and new annotated regions50 and specific regulons51. Multiple
lay-ers of regulation combined with condition-specific regulatory features52,53
allow specialized and flexible regulation of gene expression.
Additionally, quantitative transcriptomics facilitates genome-wide differential analysis of gene expression. The precise measurement of transcript abundance facilitates the discovery of the effect of gene deple-tion54,55, the elucidation of stress response56 and the mapping of detailed
expression of pathogenic islands57. In particular, Westermann et al
pro-posed a simultaneous approach to measure host and pathogen
transcrip-tomics during infection in a thought experiment58. Later, the approach
elucidated a bacterial sRNA important for intracellular survival59. The
approach has also been expanded into whole infected organ60 therefore
highlighting individual host responding to a pathogen. Finally, the com-bination of shifting paradigm into holistic approach, the availability of ( sequencing) technology and the discovery of complex bacterial gene reg-ulations specific to its niche has hastened renewed interest in the explora-tion of the prokaryotic transcriptome.
Subtlety and complexity of the pneumococcal
transcriptome
Pathogenicity island usually hosts genomic potential of pathogenic bac-teria including toxins and other virulence factors. Unfortunately, the de-termination of pathogenicity islands in S. pneumoniae has been proven
to be impossible61–63. For example, the capsule-encoding cps operon, a
well-described pneumococcal virulence factor, is conserved in both clin-ical strains of S. pneumoniae and other closely related non-pathogenic
Streptococcal species64. The presence of genes and clusters of genes, it
seems, does not determine pneumococcal virulence or pathogenicity. On the contrary, pneumococcal virulence might be determined by more subtle mutations that allow pneumococcus to precisely regulate the
ex-pression of virulence factors in response to environmental signals65. For
example, mutations in the untranslated regions preceding (5’-UTR) and following (3’-UTR) virulence genes may modify its expression level and,
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thus, determine overall pneumococcal virulence. In addition, the mul-tiple small non-coding RNA that have been reported in S. pneumoniae
further enrich the pneumococcal regulatory repertoire66–69, including the
regulation of virulence factors.
Applying cutting edge sequencing technologies to
understand pneumococcal biology
In the following dissertation, we applied recent advances in high throughput sequencing (mostly RNA) to reveal detailed organization of genetic features and pneumococcal transcription in infection models (Fig. 1). In Chapter 2, we revisited the basic genomic information of S.
pneumoniae, strain D39, Veening lab: the sequence, the annotation and
Fig. 1. Overview of thesis. We annotated the genome of S. pneumoniae D39 genome
and defined genome-wide transcriptional units by precisely mapping start and ter-mination sites (Chapter 2). Next, we elucidated transcriptional responses in response
to wide array of conditions relevant to pneumococcal lifestyle (Chapter 3). We then
established a host-pathogen infection model (Chapter 4) which we exploited to
1
R ef er enc esthe operon architecture — effectively rendering the strain to be the most well described pneumococcal strain up to date. The strain D39 has been
a major work-horse of pneumococcal research70. Next, we generated a
compendium of pneumococcal transcriptome by exposing the strain to conditions relevant to the bacterial lifestyle of colonization and
infec-tion (Chapter 3). In the same chapter, we generated a simple yet powerful
gene network as a co-expression matrix. In Chapter 4, we established,
inter alia, an infection model which contain the pneumococcus and a live human confluent lung epithelial layer. Subsequently, we exploited a dual transcriptomics approach to the aforementioned infection model to simultaneously measure the dynamic transcriptional rewiring of
epi-thelial cells and S. pneumoniae during early infection (Chapter 5). Finally,
in Chapter 6, we summarized the findings placing them in the current
scientific context.
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